Composite arrays utilizing microspheres with a hybridization chamber

ABSTRACT

The invention relates to sensor compositions comprising a composite array of individual arrays, to allow for simultaneous processing of a number of samples. The invention further provides methods of making and using the composite arrays. The invention further provides a hybridization chamber for use with a composite array.

This application is a continuing application of U.S. Ser. Nos.60/113,968, filed Dec. 28, 1998, 09/256,943, filed Feb. 24, 1999, and ofSer. No. 09/473,904, filed Dec. 28, 1999, and PCT/US99/31022, filed Dec.28, 1999, all of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to sensor compositions comprising a compositearray of individual arrays, to allow for simultaneous processing of anumber of samples. The invention further provides methods of making andusing the composite arrays. The invention further relates to anapparatus comprising a hybridization chamber for holding compositearrays.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labeling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radioisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

Of particular use in these sensors are detection mechanisms utilizingluminescence. Recently, the use of optical fibers and optical fiberstrands in combination with light absorbing dyes for chemical analyticaldeterminations has undergone rapid development, particularly within thelast decade. The use of optical fibers for such purposes and techniquesis described by Milanovich et al., “Novel Optical Fiber Techniques ForMedical Application”, Proceedings of the SPIE 28th Annual InternationalTechnical Symposium On Optics and Electro-Optics, Volume 494, 1980;Seitz, W. R., “Chemical Sensors Based On Immobilized Indicators andFiber Optics” in C.R.C. Critical Reviews In Analytical Chemistry, Vol.19, 1988, pp. 135-173; Wolfbeis, O. S., “Fiber Optical Fluorosensors InAnalytical Chemistry” in Molecular Luminescence Spectroscopy, Methodsand Applications (S. G. Schulman, editor), Wiley & Sons, New York(1988); Angel, S. M., Spectroscopy 2 (4):38 (1987); Walt, et al.,“Chemical Sensors and Microinstrumentation”, ACS Symposium Series, Vol.403, 1989, p. 252, and Wolfbeis, O. S., Fiber Optic Chemical Sensors,Ed. CRC Press, Boca Raton, Fla., 1991, 2nd Volume.

More recently, fiber optic sensors have been constructed that permit theuse of multiple dyes with a single, discrete fiber optic bundle. U.S.Pat. Nos. 5,244,636 and 5,250,264 to Walt, et al. disclose systems foraffixing multiple, different dyes on the distal end of the bundle, theteachings of each of these patents being incorporated herein by thisreference. The disclosed configurations enable separate optical fibersof the bundle to optically access individual dyes. This avoids theproblem of deconvolving the separate signals in the returning light fromeach dye, which arises when the signals from two or more dyes arecombined, each dye being sensitive to a different analyte, and there issignificant overlap in the dyes' emission spectra.

U.S. Ser. Nos. 08/818,199 and 09/151,877 describe array compositionsthat utilize microspheres or beads on a surface of a substrate, forexample on a terminal end of a fiber optic bundle, with each individualfiber comprising a bead containing an optical signature. Since the beadsgo down randomly, a unique optical signature is needed to “decode” thearray; i.e. after the array is made, a correlation of the location of anindividual site on the array with the bead or bioactive agent at thatparticular site can be made. This means that the beads may be randomlydistributed on the array, a fast and inexpensive process as compared toeither the in situ synthesis or spotting techniques of the prior art.Once the array is loaded with the beads, the array can be decoded, orcan be used, with full or partial decoding occurring after testing, asis more fully outlined below.

In addition, compositions comprising silicon wafers comprising aplurality of probe arrays in microtiter plates have been described inU.S. Pat. No. 5,545,531.

SUMMARY OF THE INVENTION

In accordance with the above objects, the present invention providescomposite array compositions comprising a first substrate with a surfacecomprising a plurality of assay locations, each assay locationcomprising a plurality of discrete sites. The substrate furthercomprises a population of microspheres comprising at least a first and asecond subpopulation, wherein each subpopulation comprises a bioactiveagent. The microspheres are distributed on each of the assay locations.

In a further aspect, the invention provides composite array compositionscomprising a first substrate with a surface comprising a plurality ofassay locations and a second substrate comprising a plurality of arraylocations, each array location comprising discrete sites. Thecompositions further comprise a population of microspheres comprising atleast a first and a second subpopulation, wherein each subpopulationcomprises a bioactive agent. The microspheres are distributed on each ofthe array locations.

In an additional aspect, the present invention provides methods ofdecoding an array composition comprising providing an array compositionas outlined above, and adding a plurality of decoding binding ligands tothe composite array composition to identify the location of at least aplurality of the bioactive agents.

In a further aspect, the present invention provides methods ofdetermining the presence of one or more target analytes in one or moresamples comprising contacting the sample with a composition as outlinedherein, and determining the presence or absence of said target analyte.

In a further aspect the invention provides a hybridization chamber. Thehybridization chamber includes a base plate and a lid. A sealant islocalized between the lid and base plate to provide for an airtightseal. When a two-component array system is used, the chamber alsoincludes component ports in the lid to immobilize the array components.That is, array components are inserted through the port in the lid. Theports may include seals so that an airtight seal is maintained. Thechamber also may include clamps and alignment pins.

In a further aspect the invention provides a hybridization chamberwherein the base plate contains holes. The holes may be in a microplatearray format. In one embodiment at least two holes are joined by achannel. In one embodiment a flexible membrane is placed on the baseplate. When pressure i.e. a vacuum, is applied to the membrane, wellsform in the membrane at the location of the holes in the base plate. Theapparatus also includes a pneumatic device for the delivery of a vacuumor positive pressure to the membrane.

In a further aspect the invention provides a method of mixing samples inan array format. The method includes providing a vacuum to the membranesuch that wells are formed. A solution is then applied to the membranesuch that at least one of the wells is filled with liquid. Subsequently,the vacuum is applied intermittently to the membrane, which results inmixing of the liquid.

In a further aspect the invention provides an apparatus comprising ahybridization chamber as described herein and any of the composite arraycompositions described herein.

In a further aspect the invention provides performing methods ofdecoding an array composition as described herein in a hybridizationchamber as described herein.

In a further aspect the invention provides performing methods ofdetermining the presence of one or more target analytes in one or moresamples as described herein in a hybridization chamber as describedherein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D and 1E depict several different “two component”system embodiments of the invention. In FIG. 1A, a bead array isdepicted. The first substrate 10 has array locations 20 with wells 25and beads 30. The second substrate 40 has assay locations 45. Anoptional lens or filter 60 is also shown; as will be appreciated bythose in the art, this may be internal to the substrate as well. FIG. 1Bis similar except that beads are not used; rather, array locations 20have discrete sites 21, 22, 23, etc. that may be formed using spotting,printing, photolithographic techniques, etc. FIGS. 1C-F depict the useof a plurality of first substrates. FIG. 1C depicts a “bead of beads”that may have additional use for mixing functions. FIG. 1D depicts aplurality of bead arrays and FIG. 1E depicts a plurality of non-beadarrays. FIG. 1F depicts the use of binding functionalities to “target”first substrates 10 to locations on the second substrate 40; as will beappreciated by those in the art, this may be done on flat secondsubstrates or on compartmentalized second substrates. FIG. 1F utilizesbinding ligand pairs 70/70′, 71/71′, 72/72′, etc. These may be eitherchemical functionalities or biological ones, such as are described forIBL/DBL pairs, such as oligonucleotides, etc.

FIGS. 2A and 2B depict two different “one component” systems. FIG. 2Adepicts a bead array, with the substrate 50 having assay locations 45with wells 25 comprising beads 30. FIG. 2B depicts a non-bead array;each assay location 45 has discrete sites 21, 22, 23, etc.

FIG. 3 depicts clustering in hyperspectral alpha space (α₁=I₁/ΣI_(I),α₂=I₂/ΣI_(i), α₃=I₃/ΣI_(i), etc.). A set of 128 different bead typespresent on a fiber bundle were decoded with by hybridizing set ofcomplementary oligonucleotides labeled with four dyes: Bodipy-493,Bodipy-R6G, Bodipy-TXR, and Bod-564 (only one dye per oligonucleotide).Shown is the second stage of a four stage decode in which 4013 beadswere decoded. Ovals are drawn around zones of hue clusters.

FIG. 4 Illustrates a two color decoding process wherein eitherFAM-labeled or Cy3-labeled oligo complements are use to “paint” (label)the different bead types on the array.

FIG. 5 depicts the decoding 128 different bead types with four colorsand four decode stages. (inset shows a single decode stage using fourdifferent dyes to decode 16 bead types.)

FIG. 6 depicts grey scale decoding of 16 different bead types. (A)Combinatorial pooling scheme for complementary decoding oligos. A (B)Two independent normalizing images were acquired, and the resulting beadintensities compared. (C) The alpha values (ratio of bead intensity inindicated decode stage to intensity in normalization image) are plottedfor three decodes stage described in (A).

FIG. 7 schematically depicts the lid and base plate. A. Depicts the lid10 and base plate 60 of the hybridization chamber. Ports 20 in the lidallow for fiber optic bundles 30 to be inserted through the lid andcontact the sample in the wells of the microtiter plate 40 in the basecavity 50 of the base plate 60. B. Depicts the base cavity 50 of thebase plate 60.

FIG. 8 schematically depicts the hybridization chamber including the lid10 and base plate 60. Also shown are the peripheral seal 80, the clamp90 and clamp receptacle 95, fiber optic bundles 30 inserted through thelid and into the well of the microtiter plate 40.

FIG. 9 depicts a base plate with holes 105. A Depicts the holes 105 inthe base plate. B Depicts channels 100 connecting the holes 105.

FIG. 10 depicts variable solution volume and localization on themembrane caused by pressure and/or vacuum. A. +P indicates pressure; −Pindicates vacuum. Upward bending of the membrane in response to pressurein all chambers and holes. B. Fluid is moved to the left side of themembrane when vacuum is applied to the left chambers and pressure isapplied to the middle and right chambers. C. When vacuum is firstapplied to the left section, fluid fills the wells. When vacuum issubsequently applied to the middle and right chambers, empty wells areformed. D. Fluid moves to the center of the membrane when vacuum isapplied to the center and pressure is applied to left and rightchambers. E. Fluid fills in wells formed by high vacuum in the center.Empty wells form on the left and right when low vacuum is applied. F.Fluid moves to the right when vacuum is applied to the right chamber andpressure is applied to the left and middle chambers.

FIG. 11 depicts a flow chart of a representative assay scheme that findsuse with the hybridization chamber.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the formation of very high densityarrays that can allow simultaneous analysis, i.e. parallel rather thanserial processing, on a number of samples. This is done by forming an“array of arrays”, i.e. a composite array comprising a plurality ofindividual arrays, that is configured to allow processing of multiplesamples. For example, each individual array is present within each wellof a microtiter plate. Thus, depending on the size of the microtiterplate and the size of the individual array, very high numbers of assayscan be run simultaneously; for example, using individual arrays of 2,000distinct species (with high levels of redundancy built in) and a 96 wellmicrotiter plate, 192,000 experiments can be done at once; the samearrays in a 384 microtiter plate yields 768,000 simultaneousexperiments, and a 1536 microtiter plate gives 3,072,000 experiments.

Generally, the array compositions of the invention can be configured inseveral ways. In a preferred embodiment, as is more fully outlinedbelow, a “one component” system is used. That is, a first substratecomprising a plurality of assay locations (sometimes also referred toherein as “assay wells”), such as a microtiter plate, is configured suchthat each assay location contains an individual array. That is, theassay location and the array location are the same. For example, theplastic material of the microtiter plate can be formed to contain aplurality of “bead wells” in the bottom of each of the assay wells.Beads containing bioactive agents can then be loaded into the bead wellsin each assay location as is more fully described below. It should benoted that while the disclosure herein emphasizes the use of beads,beads need not be used in any of the embodiments of the invention; thebioactive agents can be directly coupled to the array locations. Forexample, other types of arrays are well known and can be used in thisformat; spotted, printed or photolithographic arrays are well known; seefor example WO 95/25116; WO 95/35505; PCT US98/09163; U.S. Pat. Nos.5,700,637; 5,807,522 and 5,445,934; and U.S. Ser. Nos. 08/851,20309/187,289; and references cited within, all of which are expresslyincorporated by reference. In one component systems, if beads are notused, preferred embodiments utilize non-silicon wafer substrates.

Alternatively, a “two component” system can be used. In this embodiment,the individual arrays are formed on a second substrate, which then canbe fitted or “dipped” into the first microtiter plate substrate. As willbe appreciated by those in the art, a variety of array formats andconfigurations may be utilized. A preferred embodiment utilizes fiberoptic bundles as the individual arrays, generally with a “bead well”etched into one surface of each individual fiber, such that the beadscontaining the bioactive agent are loaded onto the end of the fiberoptic bundle. The composite array thus comprises a number of individualarrays that are configured to fit within the wells of a microtiterplate. Alternatively, other types of array formats may be used in a twocomponent system. For example, ordered arrays such as those made byspotting, printing or photolithographic techniques can be placed on thesecond substrate as outlined above. Furthermore, as shown in FIGS. 1C-F,“pieces” of arrays, either random or ordered, can be utilized as thefirst substrate.

The present invention is generally based on previous work comprising abead-based analytic chemistry system in which beads, also termedmicrospheres, carrying different chemical functionalities aredistributed on a substrate comprising a patterned surface of discretesites that can bind the individual microspheres. The beads are generallyput onto the substrate randomly, and thus several differentmethodologies can be used to “decode” the arrays. In one embodiment,unique optical signatures are incorporated into the beads, generallyfluorescent dyes, that could be used to identify the chemicalfunctionality on any particular bead. This allows the synthesis of thecandidate agents (i.e. compounds such as nucleic acids and antibodies)to be divorced from their placement on an array, i.e. the candidateagents may be synthesized on the beads, and then the beads are randomlydistributed on a patterned surface. Since the beads are first coded withan optical signature, this means that the array can later be “decoded”,i.e. after the array is made, a correlation of the location of anindividual site on the array with the bead or candidate agent at thatparticular site can be made. This means that the beads may be randomlydistributed on the array, a fast and inexpensive process as compared toeither the in situ synthesis or spotting techniques of the prior art.These methods are generally outlined in PCT US98/05025; PCT US98/21193;PCT US99/20914; PCT US99/14387; and U.S. Ser. Nos. 08/818,199;09/315,584; and 09/151,877, all of which are expressly incorporatedherein by reference. In addition, while the discussion herein isgenerally directed to the use of beads, the same configurations can beapplied to cells and other particles; see for example PCT US99/04473.

In these systems, the placement of the bioactive agents is generallyrandom, and thus a coding/decoding system is required to identify thebioactive agent at each location in the array. This may be done in avariety of ways, as is more fully outlined below, and generallyincludes: a) the use a decoding binding ligand (DBL), generally directlylabeled, that binds to either the bioactive agent or to identifierbinding ligands (IBLs) attached to the beads; b) positional decoding,for example by either targeting the placement of beads (for example byusing photoactivatable or photocleavable moieties to allow the selectiveaddition of beads to particular locations), or by using eithersub-bundles or selective loading of the sites, as are more fullyoutlined below; c) selective decoding, wherein only those beads thatbind to a target are decoded; or d) combinations of any of these. Insome cases, as is more fully outlined below, this decoding may occur forall the beads, or only for those that bind a particular target analyte.Similarly, this may occur either prior to or after addition of a targetanalyte.

Once the identity (i.e. the actual agent) and location of eachmicrosphere in the array has been fixed, the array is exposed to samplescontaining the target analytes, although as outlined below, this can bedone prior to or during the analysis as well. The target analytes willbind to the bioactive agents as is more fully outlined below, andresults in a change in the optical signal of a particular bead.

In the present invention, “decoding” can use optical signatures,decoding binding ligands that are added during a decoding step, or acombination of these methods. The decoding binding ligands will bindeither to a distinct identifier binding ligand partner that is placed onthe beads, or to the bioactive agent itself, for example when the beadscomprise single-stranded nucleic acids as the bioactive agents. Thedecoding binding ligands are either directly or indirectly labeled, andthus decoding occurs by detecting the presence of the label. By usingpools of decoding binding ligands in a sequential fashion, it ispossible to greatly minimize the number of required decoding steps.

Accordingly, the present invention provides composite array compositionscomprising at least a first substrate with a surface comprising aplurality of assay locations. By “array” herein is meant a plurality ofcandidate agents in an array format; the size of the array will dependon the composition and end use of the array. Arrays containing fromabout 2 different bioactive agents (i.e. different beads) to manymillions can be made, with very large fiber optic arrays being possible.Generally, the array will comprise from two to as many as a billion ormore, depending on the size of the beads and the substrate, as well asthe end use of the array, thus very high density, high density, moderatedensity, low density and very low density arrays may be made. Preferredranges for very high density arrays are from about 10,000,000 to about2,000,000,000, (with all numbers being per square centimeter) with fromabout 100,000,000 to about 1,000,000,000 being preferred. High densityarrays range about 100,000 to about 10,000,000, with from about1,000,000 to about 5,000,000 being particularly preferred. Moderatedensity arrays range from about 10,000 to about 100,000 beingparticularly preferred, and from about 20,000 to about 50,000 beingespecially preferred. Low density arrays are generally less than 10,000,with from about 1,000 to about 5,000 being preferred. Very low densityarrays are less than 1,000, with from about 10 to about 1000 beingpreferred, and from about 100 to about 500 being particularly preferred.In some embodiments, the compositions of the invention may not be inarray format; that is, for some embodiments, compositions comprising asingle bioactive agent may be made as well. In addition, in some arrays,multiple substrates may be used, either of different or identicalcompositions. Thus for example, large arrays may comprise a plurality ofsmaller substrates.

In addition, one advantage of the present compositions is thatparticularly through the use of fiber optic technology, extremely highdensity arrays can be made. Thus for example, because beads of 200 μm orless (with beads of 200 nm possible) can be used, and very small fibersare known, it is possible to have as many as 40,000-50,000 or more (insome instances, 1 million) different fibers and beads in a 1 mm² fiberoptic bundle, with densities of greater than 15,000,000 individual beadsand fibers (again, in some instances as many as 25-50 million) per 0.5cm² obtainable.

By “composite array” or “combination array” or grammatical equivalentsherein is meant a plurality of individual arrays, as outlined above.Generally the number of individual arrays is set by the size of themicrotiter plate used; thus, 96 well, 384 well and 1536 well microtiterplates utilize composite arrays comprising 96, 384 and 1536 individualarrays, although as will be appreciated by those in the art, not eachmicrotiter well need contain an individual array. It should be notedthat the composite arrays can comprise individual arrays that areidentical, similar or different. That is, in some embodiments, it may bedesirable to do the same 2,000 assays on 96 different samples;alternatively, doing 192,000 experiments on the same sample (i.e. thesame sample in each of the 96 wells) may be desirable. Alternatively,each row or column of the composite array could be the same, forredundancy/quality control. As will be appreciated by those in the art,there are a variety of ways to configure the system. In addition, therandom nature of the arrays may mean that the same population of beadsmay be added to two different surfaces, resulting in substantiallysimilar but perhaps not identical arrays.

By “substrate” or “solid support” or other grammatical equivalentsherein is meant any material that can be modified to contain discreteindividual sites appropriate for the attachment or association of beadsand is amenable to at least one detection method. As will be appreciatedby those in the art, the number of possible substrates is very large.Possible substrates include, but are not limited to, glass and modifiedor functionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, optical fiber bundles, and a variety of other polymers. Ingeneral, the substrates allow optical detection and do not themselvesappreciably fluorescese.

Generally the substrate is flat (planar), although as will beappreciated by those in the art, other configurations of substrates maybe used as well; for example, three dimensional configurations can beused, for example by embedding the beads in a porous block of plasticthat allows sample access to the beads and using a confocal microscopefor detection. Similarly, the beads may be placed on the inside surfaceof a tube, for flow-through sample analysis to minimize sample volume.Preferred substrates include optical fiber bundles as discussed below,and flat planar substrates such as glass, polystyrene and other plasticsand acrylics. In some embodiments, silicon wafer substrates are notpreferred. In one embodiment the substrate is in the shape of or is amicroscope slide.

The first substrate comprises a surface comprising a plurality of assaylocations, i.e. the location where the assay for the detection of atarget analyte will occur. The assay locations are generally physicallyseparated from each other, for example as assay wells in a microtiterplate, although other configurations (hydrophobicity/hydrophilicity,etc.) can be used to separate the assay locations.

In a preferred embodiment, the second substrate is an optical fiberbundle or array, as is generally described in U.S. Ser. Nos. 08/944,850and 08/519,062, PCT US98/05025, and PCT US98/09163, all of which areexpressly incorporated herein by reference. Preferred embodimentsutilize preformed unitary fiber optic arrays. By “preformed unitaryfiber optic array” herein is meant an array of discrete individual fiberoptic strands that are co-axially disposed and joined along theirlengths. The fiber strands are generally individually clad. However, onething that distinguished a preformed unitary array from other fiberoptic formats is that the fibers are not individually physicallymanipulatable; that is, one strand generally cannot be physicallyseparated at any point along its length from another fiber strand.

However, in some “two component” embodiments, the second substrate isnot a fiber optic array.

In a preferred embodiment, the assay locations (of the “one componentsystem”) or the array locations (of the “two component system”) comprisea plurality of discrete sites. Thus, in the former case, the assaylocation is the same as the array location, as described herein. In thelatter case, the array location is fitted into the assay locationseparately. In these embodiments, at least one surface of the substrateis modified to contain discrete, individual sites for later associationof microspheres (or, when microspheres are not used, for the attachmentof the bioactive agents). These sites may comprise physically alteredsites, i.e. physical configurations such as wells or small depressionsin the substrate that can retain the beads, such that a microsphere canrest in the well, or the use of other forces (magnetic or compressive),or chemically altered or active sites, such as chemically functionalizedsites, electrostatically altered sites, hydrophobically/hydrophilicallyfunctionalized sites, spots of adhesive, etc.

The sites may be a pattern, i.e. a regular design or configuration, orrandomly distributed. A preferred embodiment utilizes a regular patternof sites such that the sites may be addressed in the X-Y coordinateplane. “Pattern” in this sense includes a repeating unit cell,preferably one that allows a high density of beads on the substrate.However, it should be noted that these sites may not be discrete sites.That is, it is possible to use a uniform surface of adhesive or chemicalfunctionalities, for example, that allows the attachment of beads at anyposition. That is, the surface of the substrate is modified to allowattachment of the microspheres at individual sites, whether or not thosesites are contiguous or non-contiguous with other sites. Thus, thesurface of the substrate may be modified such that discrete sites areformed that can only have a single associated bead, or alternatively,the surface of the substrate is modified and beads may go down anywhere,but they end up at discrete sites.

In a preferred embodiment, the surface of the substrate is modified tocontain wells, i.e. depressions in the surface of the substrate. Thismay be done as is generally known in the art using a variety oftechniques, including, but not limited to, photolithography, stampingtechniques, molding techniques and microetching techniques. As will beappreciated by those in the art, the technique used will depend on thecomposition and shape of the substrate. When the first substratecomprises both the assay locations and the individual arrays, apreferred method utilizes molding techniques that form the bead wells inthe bottom of the assay wells in a microtiter plate. Similarly, apreferred embodiment utilizes a molded second substrate, comprising“fingers” or projections in an array format, and each finger comprisesbead wells.

In a preferred embodiment, physical alterations are made in a surface ofthe substrate to produce the sites. In a preferred embodiment, forexample when the second substrate is a fiber optic bundle, the surfaceof the substrate is a terminal end of the fiber bundle, as is generallydescribed in Ser. Nos. 08/818,199 and 09/151,877, both of which arehereby expressly incorporated by reference. In this embodiment, wellsare made in a terminal or distal end of a fiber optic bundle comprisingindividual fibers. In this embodiment, the cores of the individualfibers are etched, with respect to the cladding, such that small wellsor depressions are formed at one end of the fibers. The required depthof the wells will depend on the size of the beads to be added to thewells.

Generally in this embodiment, the microspheres are non-covalentlyassociated in the wells, although the wells may additionally bechemically functionalized as is generally described below, cross-linkingagents may be used, or a physical barrier may be used, i.e. a film ormembrane over the beads.

In a preferred embodiment, the surface of the substrate is modified tocontain modified sites, particularly chemically modified sites, that canbe used to attach, either covalently or non-covalently, the microspheresof the invention to the discrete sites or locations on the substrate.“Chemically modified sites” in this context includes, but is not limitedto, the addition of a pattern of chemical functional groups includingamino groups, carboxy groups, oxo groups and thiol groups, that can beused to covalently attach microspheres, which generally also containcorresponding reactive functional groups; the addition of a pattern ofadhesive that can be used to bind the microspheres (either by priorchemical functionalization for the addition of the adhesive or directaddition of the adhesive); the addition of a pattern of charged groups(similar to the chemical functionalities) for the electrostaticattachment of the microspheres, i.e. when the microspheres comprisecharged groups opposite to the sites; the addition of a pattern ofchemical functional groups that renders the sites differentiallyhydrophobic or hydrophilic, such that the addition of similarlyhydrophobic or hydrophilic microspheres under suitable experimentalconditions will result in association of the microspheres to the siteson the basis of hydroaffinity. For example, the use of hydrophobic siteswith hydrophobic beads, in an aqueous system, drives the association ofthe beads preferentially onto the sites.

In addition, biologically modified sites may be used to attach beads tothe substrate. For example, binding ligand pairs as are generallydescribed herein may be used; one partner is on the bead and the otheris on the substrate. Particularly preferred in this embodiment arecomplementary nucleic acid strands and antigen/antibody pairs.

Furthermore, the use of biological moieties in this manner allows thecreation of composite arrays as well. This is analogous to the systemdepicted in FIG. 1F, except that the substrate 10 is missing. In thisembodiment, populations of beads comprise a single binding partner, andsubpopulations of this population have different bioactive agents. Byusing different populations with different binding partners, and asubstrate comprising different assay or array locations with spatiallyseparated binding partners, a composite array can be generated. Thisembodiment also a reuse of codes, as generally described below, as eachseparate array of the composite array may use the same codes.

As outlined above, “pattern” in this sense includes the use of a uniformtreatment of the surface to allow attachment of the beads at discretesites, as well as treatment of the surface resulting in discrete sites.As will be appreciated by those in the art, this may be accomplished ina variety of ways.

As will be appreciated by those in the art, there are a number ofpossible configurations of the system, as generally depicted in theFigures. In addition to the standard formats described herein, a varietyof other formats may be used. For example, as shown in FIGS. 1C-1F,“pieces” of substrates may be used, that are not connected to oneanother. Again, these may be the same arrays or different arrays. Thesepieces may be made individually, or they may be made as a large unit ona single substrate and then the substrate is cut or separated intodifferent individual substrates. Thus, for example, FIGS. 1C and 1Ddepict a plurality of bead arrays that are added to the wells of thesecond substrate; FIG. 1C is a “bead of beads” that is configured tomaximize mixing. FIG. 1D utilizes a plurality of planar firstsubstrates; as will be appreciated by those in the art, these may or maynot be attached to the second substrate. In one embodiment, noparticular attachment means are used; alternatively, a variety ofattachment techniques are used. For example, as outlined for attachmentof beads to substrates, covalent or non-covalent forces may be used,including the use of adhesives, chemistry, hydrophobic/hydrophilicinteractions, etc. In addition, the substrate may be magnetic and heldin place (and optionally mixed) magnetically as well. Thus, for example,as depicted in FIG. 1F, binding moieties can be used; these can becovalent linkages or non-covalent linkages. They may be used simply forattachment, or for targeting the first substrate arrays to particularlocations in or on the second substrate. Thus, for example, differentoligonucleotides may be used to target and attach the first substrate tothe second.

In a preferred embodiment, there are optical properties built into thesubstrate used for imaging. Thus, for example, “lensing” capabilitiesmay be built into the substrate, either in a one component or twocomponent system. For example, in a one component system, the bottom ofone or more of the assay locations may have unique or special opticalcomponents, such as lenses, filters, etc.

In addition, preferred embodiments utilize configurations thatfacilitate mixing of the assay reaction. For example, preferredembodiments utilize two component systems that allow mixing. That is, insome embodiments, the arrays project from the block and can be used as a“stick” that stirs the reaction to facilitate good mixing of the assaycomponents, increase the kinetics of the reaction, etc. As will beappreciated by those in the art, this may be accomplished in a varietyof ways. In a preferred embodiment, the first and second substrates areconfigured such that they can be moved relative to one another, eitherin the X-Y coordinate plane, the X-Z coordinate plane, the Y-Zcoordinate plane, or in three dimensions (X-Y-Z). Preferred embodimentsutilize a block jig that allows the block to move freely in either theplane of the plate or orthogonal to it. This is particularly useful whenthe reaction volumes are small, since standard mixing conditionsfrequently do not work well in these situations.

In addition to this, or in place of it, there may be additional mixingcomponents as part of the system. For example, there may be exogeneousmixing particles added; one embodiment for example utilizes magneticparticles, with a magnet that is moved to force mixing; for examplesmall magnetic mixing bars and magnetic stir plates may be used.

Alternatively, mixing in either one or two component systems can beaccomplished by sealing the system and shaking it using standardtechniques, optionally using mixing particles.

In a preferred embodiment, the system is configured to reduceevaporation and facilitate small sample size and handling. That is, thesystem is closed or sealed by enclosing a defined space to maintaincontrol over the small sample volumes. In this regard the inventionprovides a hybridization chamber that encompasses or encloses the arrayand/or sample. As is more fully outlined below, preferred embodimentsutilize the hybridization chambers comprising a base plate and alignmentmoieties that find particular use in the two-component system, althoughthey also find use in the one-component system.

One advantage of the enclosed system is that it reduces or dampensvibration. That is, because of the small sample volume, the arrays maybe susceptible to disturbances caused by vibration, for example, byplatform shaking, motor vibration, or air circulation. By enclosing thearray, and placing the array on the base plate, the samples and arraysare less susceptible to disturbances caused by vibration as the baseplate dampens the vibration.

An additional advantage of this aspect of the invention is that theenclosed array allows for the use of increasingly small volumes. In anopen array format, small sample volumes may evaporate resulting in avariety of problems including sample variation, alteration andinconsistent concentration of solutes in the solution. For example, whensmall sample volumes are present in different assay locations,differential evaporation of the solution may result in dramaticallyaltered solute concentration. Such differences may alter hybridizationkinetics, for example, and make it difficult to interpret and compareresults obtained from such open arrays. However, by enclosing the array,for example in the hybridization chamber outlined herein, such samplevariance is minimized thereby rendering the data obtained from theenclosed array more reliable. Accordingly, any of the methods describedherein, find use with the hybridization chamber.

Also, the enclosed array allows for prolonged assay/incubation timesrelative to incubation times in an open array. Again, the sealed orenclosed array prevents sample evaporation, allowing for prolongedincubation periods.

In addition, the enclosed array facilitates mixing of the sample, whennecessary. In general, when using small sample volumes, adequate mixingof the sample may be difficult to achieve. However, as is more fullyoutlined below, in one embodiment the hybridization chamber facilitatesmixing when flexible membranes are used with a pneumatic device thatprovides vacuum and/or pressure.

When a “two-component” system is used, a hybridization chamber may beused. That is, both of the components i.e. the substrate comprising aplurality of assay locations and the substrate comprising a plurality ofarray locations, are enclosed within the hybridization chamber. In apreferred embodiment, these components include but are not limited to afiber optic array and a multi-well microtiter plate that are enclosed inthe hybridization chamber.

In a preferred embodiment the hybridization chamber contains a baseplate upon which or into which one of the components is placed. By baseplate is meant any platform or holder onto which one of the arraycomponents is placed. The base plate may be made of any materialincluding plastic, glass or metal or any materials outlined herein forsubstrates; when the base plate is metal, it is preferably made ofaluminum. Aluminum provides for a light weight yet durable base plate.In addition, aluminum allows for efficient and/or rapid heat transfer tothe chamber. However, when the base plate is made of plastic or glass,the component is directly contacted with the base plate. Alternatively,metal sheets or templates may be inserted into or placed on the baseplate. The metal sheets or templates can be designed to hold any of avariety of shapes to accommodate a variety of component sizes and/orshapes. As previously described, metal offers the advantage of being arapid and efficient heat conductor.

In one embodiment the base plate contains at least one depression orbase cavity into which the array component is placed. That is, when amicrotiter plate is the component, for example, the depression or basecavity is formed such that the microtiter plate is placed directly intoit and preferably fits tightly to avoid extra vibration and allowefficient heat transfer. The depression may be molded into the baseplate. In addition, the base plate may contain multiple depressions orcavities such that multiple separate array components are placed on asingle base plate. Alternatively, the base plate may be flat, andpreferably comprise hooks or other attachment moieties to keep thearrays in place.

In addition preferred embodiments utilize a lid with the hybridizationchamber. The lid can be made of any material (again, as listed forsubstrates herein), but glass, plastics or metal is preferred. The lidis preferably matched to the base plate such that when the lid is placedon the base plate, a closed chamber is formed.

In another embodiment the lid comprises at least one component placementport. By component placement port is meant a site in the lid to which acomponent is immobilized. That is, the placement port allows forattachment of one of the components to the lid. In a preferredembodiment, the port is a hole in the lid through which the component isinserted. For example, when a fiber optic bundle is the component, thebundle is inserted through the port. In this embodiment, the portadditionally comprises a sealant surrounding the attachment site, suchthat an airtight seal is formed between the component, i.e. the distalend of the fiber optic bundle, and the lid. This sealant may be anymaterial including silicon, rubber, plastic, etc., as outlined below.Alternatively, the seal may be a gel-based substance such as petroleumjelly, or a film based substance such as PARAFILM.

In an additional embodiment, the lid comprises a plurality of ports inthe lid. That is, when multiple components are to be used, it isnecessary to have a separate port for each component. For example, whenmultiple fiber optic bundles are used, each fiber optic bundle is placedin a separate port. However, although it is possible for each fiberoptic bundle to be inserted into one port at a time, it is also possiblefor the same fiber optic bundle to be inserted into different portssuccessively. That is, there is nothing to limit the number of portsinto which a component is inserted successively. For example, as shownin FIG. 7A the lid 10 contains multiple ports 20 into which fiber opticbundles 30 are placed. The lid is then placed onto a microtiter plate 40in the base cavity 50 of the base plate 60. A base plate 60 is depictedin FIG. 7 b and shows the base plate 60 and base cavity 50.

In a preferred embodiment, the port seal reduces or prevents solutioncross contamination. That is, the seal surrounding the individualport/component forms a seal against the base plate or array componentsuch that the solution from the sample corresponding to a particularport/component is separated or sealed from the other components.

In an alternative embodiment, not all ports are filled with componentsat all times. When it is appropriate or desired to have less thanmaximal filling of the ports, plugs can be inserted into the ports thatdo not contain components. In this manner, the lid still forms anairtight seal with the base plate, despite the presence of ports withoutcomponents. The plugs can be in the form of a rubber stopper, a gasket,a film, a gel and the like.

In a preferred embodiment around the periphery of the chamber betweenthe lid and base plate resides a sealant. The sealant may be of anymaterial that results in an airtight seal being formed between the lidand base plate. In a preferred embodiment, the sealant is formed ofrubber, such as a rubber or silicon gasket or O-ring 80 (see FIG. 8).The sealant may be fixed to either the lid or baseplate. To this end,the sealant may be permanently affixed to the lid or baseplate.Alternatively, the sealant may fit into a groove in either the lid orbase plate. As such, the sealant is immobilized to the lid or baseplate, but the immobilization is not necessarily permanent.Alternatively, the sealant may be formed from a liquid sealant such aspetroleum jelly or from a pliable film material such as PARAFILM orother waxes.

In a preferred embodiment, when a two-component system is used, thehybridization chamber further comprises alignment moieties. By alignmentmoieties is meant a feature of the chamber that facilitates alignment ofthe lid with the base plate. The importance of the alignment moietiesresides not only in the alignment of a single lid and base plate, butalso reproducible alignment of multiple lids and base plates. That is,the alignment moieties facilitate the physical alignment between anyarray components and any multiple well microtiter plate configuration.When fiber optic bundles in the lid are to be aligned with a microtiterplate on the base plate, the alignment moieties allow for alignment ofthe vertical center axis of the fiber bundle with their correspondingwell center axis. In a preferred embodiment, alignment is such that allfiber bundles clear, i.e. do not touch, the inner walls of the wells.This alignment may be important for sequential imaging.

In one embodiment the alignment moiety is a complementary male/femalefitting. The male fitting may be affixed to the lid or base plate,although it need not be permanently affixed. When a male fitting is usedas an alignment moiety in either the base plate or lid of the chamber,it is preferable that the opposite chamber piece contain a slot or hole(female fitting) into which the male fitting is inserted. One ofordinary skill in the art appreciates the variations of this male/femalefitting that find use with the invention. In this regard, the featuresmay be indexer pins or bumps on one chamber piece and holes orcomplementary grooves on the other piece.

In a preferred embodiment, fiducials are used; see U.S. Ser. Nos.60/119,323, and 09/500,555 and PCT/US00/03375, hereby incorporated byreference in their entirety.

In an alternative embodiment, the chamber may also contain clampfeatures to maintain secure contact between the lid and base plate. Theadvantage of clamping is to distribute uniform loading throughout thechamber to accomplish uniform seal compression. By “clamp features” or“clamps” is meant any feature that allows for the application andmaintenance of increased pressure or a seal between the lid and baseplate. In one embodiment, the claim feature includes a rotatingstud/receptacle mechanism. That is, a stud 90 is inserted into areceptacle 95 and rotated to depress the lid and base plate together(see FIG. 8). Alternatively, the mechanism may include a hook and latchmechanism. One of ordinary skill in the art appreciates the number ofclamping mechanisms that find use with the invention. In addition, oneof ordinary skill in the art appreciates that the method of clamping isnot limited to manual clamping. As such, it may also be automated.

In an alternative embodiment, the chamber includes features around theperiphery for handling the chamber. In a preferred embodiment thefeatures are slots that are wide enough to permit a users fingers tomanually handle the chamber/array. In an alternative embodiment, thefeatures are slots, grooves, handles and the like and may findparticular use in automatic or robotic movement of the chambers. Theseadditional features may also be distributed asymmetrically to facilitaterobotic handling.

As described above, an advantage of the hybridization chamber is thatsmall sample volumes can be used without the loss of sample solution. Ina further embodiment, the chamber may contain one or more reservoirs tohold additional solutions. As such, the hybridization chamber alsofunctions as a humidity chamber. The inclusion of additional solution inthe reservoir further prevents evaporation of sample.

In an alternative embodiment, for example when no microtiter plate isused, the sample may be applied to a membrane that is on the surface ofa base plate. Advantages of using the membrane include ease of cleaningor even disposing of the membrane after each use and the flexiblemembrane will not damage pipette tips or fiber optic tips due tocontacting the tips with the bottom of the sample well.

In this embodiment, the base plate contains a series of small openings105, for example in microplate format (FIG. 9A). Thus, the membrane isdepressed into the openings forming separate assay locations. A varietyof membranes are useful with the invention. What is important is thatthe membrane is flexible. In some embodiments it may be desired to havea chemically inert membrane, while in some embodiments it may bedesirable to have a membrane to which assay components will interact,for example nylon, nitrocellulose membranes and the like.

In a preferred embodiment, channels connect each of the openings (FIG.9B). The channels 100 may connect to a pneumatic device that producesvacuum and/or pressure. Thus, when vacuum is applied, the membranedeforms into the openings 105 to form small pockets or wells. The samplecan then be applied to the pockets. By applying different amounts ofvacuum to the membrane through the openings, the volume of the wellformed by the deformed membrane and fluid height can be changed.Furthermore, applying intermittent vacuum to the membrane through thechannel can also agitate or mix the liquid in the wells. Such a mixingmethod is advantageous because the entire system does not have to bevibrated and stir bars or tumblers are not required. Furthermore, whensubsets of openings are connected to different channels, differentsubsets can be mixed independently in the same base plate.

When positive pressure is applied, the membrane deforms up or stays flatdepending on the magnitude of the pressure, whether there is a load ontop of the membrane and the size and shape of the opening. This hassignificant advantages particularly in washing or cleaning of thechamber.

When pressure and vacuum are applied to different ports in certainsequences, small amounts of solutions can be made to migrate todifferent portions of the membrane. That is, as shown in FIG. 10A-F,differential application of pressure and vacuum results in a membranethat is elevated in some places and depressed in other places. Thus, asolution that is applied to the membrane will migrate to the lowersections of the membrane. This has the advantage of allowing incubationsof a sample on the membrane to proceed for precise times. That isfollowing the particular time, vacuum can be released and if necessarypressure applied to remove the solution. This will allow the incubationin small sections to achieve uniform incubation time between the firstand last wells across an array.

Advantages of regulating sample volume through the application of vacuumor pressure, include reducing consumption volume of reagents, such ashybridization solutions; increasing the ease of mixing small samplevolumes and increasing the ease of cleaning the membrane.

In a preferred embodiment the channels connect to common fluid handlingdevices to pump in or suck out sample solutions such as hybridizationmixtures or wash fluids. Again, in one embodiment all openings areconnected to a single channel. As such, all wells are treated with thesame solution. Alternatively, subpopulations of openings are connectedto different channels allowing for differential application of solutionsto the subpopulations.

When the channels are connected to fluid handling devices, it will benecessary to include a feature for the application and removal of theliquid from the sample. That is, for liquid to be added and removedthrough the opening in the base plate, the membrane must be penetratedto allow the fluid to be moved. In this regard, a needle, for example,is useful for puncturing the membrane to apply and remove the fluid.When needles are used, it may be necessary to use a resealable membrane,or apply a sealant to the puncture location to prevent undesired leakageof the solution.

In some embodiments the chamber includes heat transfer features. Thatis, when elevated temperatures are required or desired, the chamber isdesigned to maintain elevated temperatures. In one embodiment, thisincludes the application of an insulating material to the chamber. Then,when pre-warmed solution is introduced into the chamber, the elevatedtemperature is maintained. That is, the solution can be easily heatedoutside of the chamber prior to being pumped into the chamber. Thesimple chamber geometry will facilitate the maintenance of equaltemperatures between liquid in different wells.

In an alternative embodiment, the chamber includes a heating mechanismto maintain the elevated temperature in the chamber. In one embodiment,the chamber is heated uniformly by the heating apparatus. In analternative embodiment, the heating apparatus heats different sectionsof the chamber independently.

As described above, the use of metal such as aluminum on the base platefacilitates heat transfer because the metal is a fast and efficientconductor of heat.

When a “one-component” system is used, a lid and a sealing mechanism canbe used. That is, as described above, the lid forms an airtight sealwith the base plate. Thus, like the lid above, the lid of the“one-component” system also includes a sealant between the lid and baseplate. In one embodiment, the lid and base plate also include alignmentmoieties as described above for the “two-component” system.Alternatively, in one embodiment the chamber of the one-component systemdoes not include alignment moieties. In this respect, the necessity forstringent alignment of the lid and base plate in the one-componentsystem is lower than that for the two-component system. That is, becausethe one-component system does not have array components in the lid to bealigned with array locations on the base plate, alignment is not asstringent. However, alignment may still be important for imaging.

Furthermore, as described above, the lid of the chamber in theone-component system can be made of glass, plastic or metal. Again, theuse of metal facilitates the maintenance of temperature as the metal isa fast and efficient heat conductor.

In addition, the system may comprise additional elements as well. Theseinclude a holder or holders for the probes or fiber optic bundles. Suchholders are more fully described in U.S. Ser. Nos. 60/135,089, filed May20, 1999, and 09/574,962 filed May 19, 2000, and PCT US00/13772 filedMay 19, 2000. In addition, the system may include cells as described inU.S. Ser. Nos. 09/033,462 and 09/260,963 and PCT/US99/04473. Inaddition, the system may include fiducials as described in U.S. Ser.Nos. 60/119,323, and 09/500,555 and PCT/US00/03375, all of which areexpressly incorporated herein by reference.

In a preferred embodiment, the methods and compositions of the inventioncomprise a robotic system. Many systems are generally directed to theuse of 96 (or more) well microtiter plates, but as will be appreciatedby those in the art, any number of different plates or configurationsmay be used. In addition, any or all of the steps outlined herein may beautomated; thus, for example, the systems may be completely or partiallyautomated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates;automated lid handlers to remove and replace lids for wells on non-crosscontamination plates; tip assemblies for sample distribution withdisposable tips; washable tip assemblies for sample distribution; 96well loading blocks; cooled reagent racks; microtiter plate pipettepositions (optionally cooled); stacking towers for plates and tips; andcomputer systems.

Fully robotic systems include automated liquid- and particle-handing,including high throughput pipetting to perform all steps of screeningapplications. This includes liquid, and particle manipulations such asaspiration, dispensing, mixing, diluting, washing, accurate volumetrictransfers; retrieving, and discarding of pipette tips; and repetitivepipetting of identical volumes for multiple deliveries from a singlesample aspiration. These manipulations are cross-contamination-freeliquid and particle transfers.

In a preferred embodiment, chemically derivatized particles, plates,tubes, magnetic particle, or other solid phase matrix with specificityto the ligand or variant proteins are used. The binding surfaces ofmicroplates, tubes or any solid phase matrices include non-polarsurfaces, highly polar surfaces, modified dextran coating to promotecovalent binding, antibody coating, affinity media to bind fusionproteins or peptides, surface-fixed proteins such as recombinant proteinA or G, nucleotide resins or coatings, and other affinity matrix areuseful in this invention.

In a preferred embodiment, platforms for multi-well plates, multi-tubes,minitubes, deep-well plates, microfuge tubes, cryovials, square wellplates, filters, chips, optic fibers, beads, and other solid-phasematrices or platform with various volumes are accommodated on anupgradeable modular platform for additional capacity. This modularplatform includes a variable speed orbital shaker, and multi-positionwork decks for source samples, sample and reagent dilution, assayplates, sample and reagent reservoirs, pipette tips, and an active washstation.

In a preferred embodiment, thermocycler and thermoregulating systems areused for stabilizing the temperature of the heat exchangers such ascontrolled blocks or platforms to provide accurate temperature controlof incubating samples from 4° C. to 100° C.

In a preferred embodiment, Interchangeable pipette heads (single ormulti-channel) with single or multiple magnetic probes, affinity probes,or pipetters robotically manipulate the liquid and particles. Multi-wellor multi-tube magnetic separators or platforms manipulate liquid andparticles in single or multiple sample formats.

In some preferred embodiments, the instrumentation will include CCDcameras to capture and transform data and images into quantifiableformats; and a computer workstation. These will enable data analysis.

The flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.The customized tools, labware, and liquid and particle transfer patternsallow different applications to be performed. The database allows methodand parameter storage. Robotic and computer interfaces allowcommunication between instruments.

In a preferred embodiment, the robotic workstation includes one or moreheating or cooling components. Depending on the reactions and reagents,either cooling or heating may be required, which can be done using anynumber of known heating and cooling systems, including Peltier systems.

In a preferred embodiment, the robotic apparatus includes a centralprocessing unit which communicates with a memory and a set ofinput/output devices (e.g., keyboard, mouse, monitor, printer, etc.)through a bus. The general interaction between a central processingunit, a memory, input/output devices, and a bus is known in the art.Thus, a variety of different procedures, depending on the experiments tobe run, are stored in the CPU memory.

In a preferred embodiment, the compositions of the invention furthercomprise a population of microspheres. By “population” herein is meant aplurality of beads as outlined above for arrays. Within the populationare separate subpopulations, which can be a single microsphere ormultiple identical microspheres. That is, in some embodiments, as ismore fully outlined below, the array may contain only a single bead foreach bioactive agent; preferred embodiments utilize a plurality of beadsof each type.

By “microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. The composition of the beadswill vary, depending on the class of bioactive agent and the method ofsynthesis. Suitable bead compositions include those used in peptide,nucleic acid and organic moiety synthesis, including, but not limitedto, plastics, ceramics, glass, polystyrene, methylstyrene, acrylicpolymers, paramagnetic materials, thoria sol, carbon graphite, titaniumdioxide, latex or cross-linked dextrans such as Sepharose, cellulose,nylon, cross-linked micelles and Teflon may all be used. “MicrosphereDetection Guide” from Bangs Laboratories, Fishers Ind. is a helpfulguide.

The beads need not be spherical; irregular particles may be used. Inaddition, the beads may be porous, thus increasing the surface area ofthe bead available for either bioactive agent attachment or IBLattachment. The bead sizes range from nanometers, i.e. 100 nm, tomillimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200microns being preferred, and from about 0.5 to about 5 micron beingparticularly preferred, although in some embodiments smaller beads maybe used.

It should be noted that a key component of the invention is the use of asubstrate/bead pairing that allows the association or attachment of thebeads at discrete sites on the surface of the substrate, such that thebeads do not move during the course of the assay.

Each microsphere comprises a bioactive agent, although as will beappreciated by those in the art, there may be some microspheres which donot contain a bioactive agent, depending on the synthetic methods. By“candidate bioactive agent” or “bioactive agent” or “chemicalfunctionality” or “binding ligand” herein is meant as used hereindescribes any molecule, e.g., protein, oligopeptide, small organicmolecule, coordination complex, polysaccharide, polynucleotide, etc.which can be attached to the microspheres of the invention. It should beunderstood that the compositions of the invention have two primary uses.In a preferred embodiment, as is more fully outlined below, thecompositions are used to detect the presence of a particular targetanalyte; for example, the presence or absence of a particular nucleotidesequence or a particular protein, such as an enzyme, an antibody or anantigen. In an alternate preferred embodiment, the compositions are usedto screen bioactive agents, i.e. drug candidates, for binding to aparticular target analyte.

Bioactive agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 Daltons.Bioactive agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The bioactiveagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Bioactive agents are also found amongbiomolecules including peptides, nucleic acids, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof. Particularly preferred are nucleic acids andproteins.

Bioactive agents can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means. Knownpharmacological agents may be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification and/oramidification to produce structural analogs.

In a preferred embodiment, the bioactive agents are proteins. By“protein” herein is meant at least two covalently attached amino acids,which includes proteins, polypeptides, oligopeptides and peptides. Theprotein may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. Thus “amino acid”, or“peptide residue”, as used herein means both naturally occurring andsynthetic amino acids. For example, homo-phenylalanine, citrulline andnorleucine are considered amino acids for the purposes of the invention.The side chains may be in either the (R) or the (S) configuration. Inthe preferred embodiment, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradations.

In one preferred embodiment, the bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eukaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the bioactive agents are peptides of fromabout 5 to about 30 amino acids, with from about 5 to about 20 aminoacids being preferred, and from about 7 to about 15 being particularlypreferred. The peptides may be digests of naturally occurring proteinsas is outlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized bioactive proteinaceous agents.

In a preferred embodiment, a library of bioactive agents are used. Thelibrary should provide a sufficiently structurally diverse population ofbioactive agents to effect a probabilistically sufficient range ofbinding to target analytes. Accordingly, an interaction library must belarge enough so that at least one of its members will have a structurethat gives it affinity for the target analyte. Although it is difficultto gauge the required absolute size of an interaction library, natureprovides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity forthe target. Thus, in a preferred embodiment, at least 10⁶, preferably atleast 10⁷, more preferably at least 10⁸ and most preferably at least 10⁹different bioactive agents are simultaneously analyzed in the subjectmethods. Preferred methods maximize library size and diversity.

In a preferred embodiment, the library is fully randomized, with nosequence preferences or constants at any position. In a preferredembodiment, the library is biased. That is, some positions within thesequence are either held constant, or are selected from a limited numberof possibilities. For example, in a preferred embodiment, thenucleotides or amino acid residues are randomized within a definedclass, for example, of hydrophobic amino acids, hydrophilic residues,sterically biased (either small or large) residues, towards the creationof cysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the bioactive agents are nucleic acids(generally called “probe nucleic acids” or “candidate probes” herein).By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl,et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al., Nucl. AcidsRes., 14:3487 (1986); Sawai, et al., Chem. Lett., 805 (1984), Letsinger,et al., J. Am. Chem. Soc., 110:4470 (1988); and Pauwels, et al., ChemicaScripta, 26:141 (1986)), phosphorothioate (Mag, et al., Nucleic AcidsRes., 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate(Briu, et al., J. Am. Chem. Soc., 111:2321 (1989)),O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.,114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl., 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207(1996), all of which are incorporated by reference)). Other analognucleic acids include those with positive backbones (Denpcy, et al.,Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionic backbones (U.S.Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863;Kiedrowshi, et al., Angew. Chem. Intl. Ed. English, 30:423 (1991);Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); Letsinger, etal., Nucleosides & Nucleotides, 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, et al.,Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., (1995) pp. 169-176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments; for example, PNA is particularly preferred. In addition,mixtures of naturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made. Thenucleic acids may be single stranded or double stranded, as specified,or contain portions of both double stranded or single stranded sequence.The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid,where the nucleic acid contains any combination of deoxyribo- andribo-nucleotides, and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, inosine, xanthanine,hypoxanthanine, isocytosine, isoguanine, and base analogs such asnitropyrrole and nitroindole, etc.

In a preferred embodiment, the bioactive agents are libraries of clonalnucleic acids, including DNA and RNA. In this embodiment, individualnucleic acids are prepared, generally using conventional methods(including, but not limited to, propagation in plasmid or phage vectors,amplification techniques including PCR, etc.). The nucleic acids arepreferably arrayed in some format, such as a microtiter plate format,and beads added for attachment of the libraries.

Attachment of the clonal libraries (or any of the nucleic acids outlinedherein) may be done in a variety of ways, as will be appreciated bythose in the art, including, but not limited to, chemical or affinitycapture (for example, including the incorporation of derivatizednucleotides such as AminoLink or biotinylated nucleotides that can thenbe used to attach the nucleic acid to a surface, as well as affinitycapture by hybridization), cross-linking, and electrostatic attachment,etc.

In a preferred embodiment, affinity capture is used to attach the clonalnucleic acids to the beads. For example, cloned nucleic acids can bederivatized, for example with one member of a binding pair, and thebeads derivatized with the other member of a binding pair. Suitablebinding pairs are as described herein for IBL/DBL pairs. For example,the cloned nucleic acids may be biotinylated (for example usingenzymatic incorporate of biotinylated nucleotides, for by photoactivatedcross-linking of biotin). Biotinylated nucleic acids can then becaptured on streptavidin-coated beads, as is known in the art.Similarly, other hapten-receptor combinations can be used, such asdigoxigenin and anti-digoxigenin antibodies. Alternatively, chemicalgroups can be added in the form of derivatized nucleotides, that canthem be used to add the nucleic acid to the surface.

Preferred attachments are covalent, although even relatively weakinteractions (i.e. non-covalent) can be sufficient to attach a nucleicacid to a surface, if there are multiple sites of attachment per eachnucleic acid. Thus, for example, electrostatic interactions can be usedfor attachment, for example by having beads carrying the opposite chargeto the bioactive agent.

Similarly, affinity capture utilizing hybridization can be used toattach cloned nucleic acids to beads. For example, as is known in theart, polyA+RNA is routinely captured by hybridization to oligo-dT beads;this may include oligo-dT capture followed by a cross-linking step, suchas psoralen crosslinking). If the nucleic acids of interest do notcontain a polyA tract, one can be attached by polymerization withterminal transferase, or via ligation of an oligoA linker, as is knownin the art.

Alternatively, chemical crosslinking may be done, for example byphotoactivated crosslinking of thymidine to reactive groups, as is knownin the art.

In general, special methods are required to decode clonal arrays, as ismore fully outlined below.

As described above generally for proteins, nucleic acid bioactive agentsmay be naturally occurring nucleic acids, random nucleic acids, or“biased” random nucleic acids. For example, digests of procaryotic oreukaryotic genomes may be used as is outlined above for proteins.

In general, probes of the present invention are designed to becomplementary to a target sequence (either the target analyte sequenceof the sample or to other probe sequences, as is described herein), suchthat hybridization of the target and the probes of the present inventionoccurs. This complementarily need not be perfect; there may be anynumber of base pair mismatches that will interfere with hybridizationbetween the target sequence and the single stranded nucleic acids of thepresent invention. However, if the number of mutations is so great thatno hybridization can occur under even the least stringent ofhybridization conditions, the sequence is not a complementary targetsequence. Thus, by “substantially complementary” herein is meant thatthe probes are sufficiently complementary to the target sequences tohybridize under the selected reaction conditions. High stringencyconditions are known in the art; see for example Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et al., both of which arehereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength pH. The T_(m) is the temperature (under defined ionic strength,pH and nucleic acid concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g. greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. In another embodiment, less stringenthybridization conditions are used; for example, moderate or lowstringency conditions may be used, as are known in the art; see Maniatisand Ausubel, supra, and Tijssen, supra.

The term “target sequence” or grammatical equivalents herein means anucleic acid sequence on a single strand of nucleic acid. The targetsequence may be a portion of a gene, a regulatory sequence, genomic DNA,cDNA, RNA including mRNA and rRNA, or others. It may be any length, withthe understanding that longer sequences are more specific. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. As is outlined morefully below, probes are made to hybridize to target sequences todetermine the presence or absence of the target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart.

In a preferred embodiment, the bioactive agents are organic chemicalmoieties, a wide variety of which are available in the literature.

In a preferred embodiment, each bead comprises a single type ofbioactive agent, although a plurality of individual bioactive agents arepreferably attached to each bead. Similarly, preferred embodimentsutilize more than one microsphere containing a unique bioactive agent;that is, there is redundancy built into the system by the use ofsubpopulations of microspheres, each microsphere in the subpopulationcontaining the same bioactive agent.

As will be appreciated by those in the art, the bioactive agents mayeither be synthesized directly on the beads, or they may be made andthen attached after synthesis. In a preferred embodiment, linkers areused to attach the bioactive agents to the beads, to allow both goodattachment, sufficient flexibility to allow good interaction with thetarget molecule, and to avoid undesirable binding reactions.

In a preferred embodiment, the bioactive agents are synthesized directlyon the beads. As is known in the art, many classes of chemical compoundsare currently synthesized on solid supports, including beads, such aspeptides, organic moieties, and nucleic acids.

In a preferred embodiment, the bioactive agents are synthesized first,and then covalently attached to the beads. As will be appreciated bythose in the art, this will be done depending on the composition of thebioactive agents and the beads. The functionalization of solid supportsurfaces such as certain polymers with chemically reactive groups suchas thiols, amines, carboxyls, etc. is generally known in the art.Accordingly, “blank” microspheres may be used that have surfacechemistries that facilitate the attachment of the desired functionalityby the user. Some examples of these surface chemistries for blankmicrospheres include, but are not limited to, amino groups includingaliphatic and aromatic amines, carboxylic acids, aldehydes, amides,chloromethyl groups, hydrazide, hydroxyl groups, sulfonates andsulfates.

These functional groups can be used to add any number of differentcandidate agents to the beads, generally using known chemistries. Forexample, candidate agents containing carbohydrates may be attached to anamino-functionalized support; the aldehyde of the carbohydrate is madeusing standard techniques, and then the aldehyde is reacted with anamino group on the surface. In an alternative embodiment, a sulfhydryllinker may be used. There are a number of sulfhydryl reactive linkersknown in the art such as SPDP, maleimides, α-haloacetyls, and pyridyldisulfides (see for example the 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference) which can be used to attach cysteine containingproteinaceous agents to the support. Alternatively, an amino group onthe candidate agent may be used for attachment to an amino group on thesurface. For example, a large number of stable bifunctional groups arewell known in the art, including homobifunctional and heterobifunctionallinkers (see Pierce Catalog and Handbook, pages 165-200). In anadditional embodiment, carboxyl groups (either from the surface or fromthe candidate agent) may be derivatized using well known linkers (seethe Pierce catalog). For example, carbodiimides activate carboxyl groupsfor attack by good nucleophiles such as amines (see Torchilin et al.,Critical Rev. Therapeutic Drug Carrier Systems, 7(4):275-308 (1991),expressly incorporated herein). Proteinaceous candidate agents may alsobe attached using other techniques known in the art, for example for theattachment of antibodies to polymers; see Slinkin et al., Bioconj. Chem.2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconj.Chem. 3:323-327 (1992); King et al., Cancer Res. 54:6176-6185 (1994);and Wilbur et al., Bioconjugate Chem. 5:220-235 (1994), all of which arehereby expressly incorporated by reference). It should be understoodthat the candidate agents may be attached in a variety of ways,including those listed above. Preferably, the manner of attachment doesnot significantly alter the functionality of the candidate agent; thatis, the candidate agent should be attached in such a flexible manner asto allow its interaction with a target. In addition, these types ofchemical or biological functionalities may be used to attach arrays toassay locations, as is depicted in FIG. 1F, or individual sets of beads.

Specific techniques for immobilizing enzymes on microspheres are knownin the prior art. In one case, NH₂ surface chemistry microspheres areused. Surface activation is achieved with a 2.5% glutaraldehyde inphosphate buffered saline (10 mM) providing a pH of 6.9. (138 mM NaCl,2.7 mM, KCl). This is stirred on a stir bed for approximately 2 hours atroom temperature. The microspheres are then rinsed with ultrapure waterplus 0.01% tween 20 (surfactant)-0.02%, and rinsed again with a pH 7.7PBS plus 0.01% tween 20. Finally, the enzyme is added to the solution,preferably after being prefiltered using a 0.45 μm amicon micropurefilter.

In some embodiments, the microspheres may additionally compriseidentifier binding ligands for use in certain decoding systems. By“identifier binding ligands” or “IBLs” herein is meant a compound thatwill specifically bind a corresponding decoder binding ligand (DBL) tofacilitate the elucidation of the identity of the bioactive agentattached to the bead. That is, the IBL and the corresponding DBL form abinding partner pair. By “specifically bind” herein is meant that theIBL binds its DBL with specificity sufficient to differentiate betweenthe corresponding DBL and other DBLs (that is, DBLs for other IBLs), orother components or contaminants of the system. The binding should besufficient to remain bound under the conditions of the decoding step,including wash steps to remove non-specific binding.

In some embodiments, for example when the IBLs and corresponding DBLsare proteins or nucleic acids, the dissociation constants of the IBL toits DBL will be less than about 10⁻⁴-10⁻⁶ M⁻¹, with less than about 10⁻⁵to 10⁻⁹ M⁻¹ being preferred and less than about 10⁻⁷-10⁻⁹ M⁻¹ beingparticularly preferred.

IBL-DBL binding pairs are known or can be readily found using knowntechniques. For example, when the IBL is a protein, the DBLs includeproteins (particularly including antibodies or fragments thereof (FAbs,etc.)) or small molecules, or vice versa (the IBL is an antibody and theDBL is a protein). Metal ion-metal ion ligands or chelators pairs arealso useful. Antigen-antibody pairs, enzymes and substrates orinhibitors, other protein-protein interacting pairs, receptor-ligands,complementary nucleic acids (including nucleic acid molecules that formtriple helices), and carbohydrates and their binding partners are alsosuitable binding pairs. Nucleic acid-nucleic acid binding proteins pairsare also useful, including single-stranded or double-stranded nucleicacid binding proteins, and small molecule nucleic acid binding agents.Similarly, as is generally described in U.S. Pat. Nos. 5,270,163,5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, andrelated patents, hereby incorporated by reference, nucleic acid“aptamers” can be developed for binding to virtually any target; such anaptamer-target pair can be used as the IBL-DBL pair. Similarly, there isa wide body of literature relating to the development of binding pairsbased on combinatorial chemistry methods.

In a preferred embodiment, the IBL is a molecule whose color orluminescence properties change in the presence of a selectively-bindingDBL.

In one embodiment, the DBL may be attached to a bead, i.e. a “decoderbead”, that may carry a label such as a fluorophore.

In a preferred embodiment, the IBL-DBL pair comprise substantiallycomplementary single-stranded nucleic acids. In this embodiment, thebinding ligands can be referred to as “identifier probes” and “decoderprobes”. Generally, the identifier and decoder probes range from about 4basepairs in length to about 1000, with from about 6 to about 100 beingpreferred, and from about 8 to about 40 being particularly preferred.What is important is that the probes are long enough to be specific,i.e. to distinguish between different IBL-DBL pairs, yet short enough toallow both a) dissociation, if necessary, under suitable experimentalconditions, and b) efficient hybridization.

In a preferred embodiment, as is more fully outlined below, the IBLs donot bind to DBLs. Rather, the IBLs are used as identifier moieties(“IMs”) that are identified directly, for example through the use ofmass spectroscopy.

Alternatively, in a preferred embodiment, the IBL and the bioactiveagent are the same moiety; thus, for example, as outlined herein,particularly when no optical signatures are used, the bioactive agentcan serve as both the identifier and the agent. For example, in the caseof nucleic acids, the bead-bound probe (which serves as the bioactiveagent) can also bind decoder probes, to identify the sequence of theprobe on the bead. Thus, in this embodiment, the DBLs bind to thebioactive agents. This is particularly useful as this embodiment cangive information about the array or the assay in addition to decoding.For example, as is more fully described below, the use of the DBLsallows array calibration and assay development. This may be done even ifthe DBLs are not used as such; for example in non-random arrays, the useof these probe sets can allow array calibration and assay developmenteven if decoding is not required.

In a preferred embodiment, the microspheres do not contain an opticalsignature. That is, as outlined in U.S. Ser. Nos. 08/818,199 and09/151,877, previous work had each subpopulation of microspherescomprising a unique optical signature or optical tag that is used toidentify the unique bioactive agent of that subpopulation ofmicrospheres; that is, decoding utilizes optical properties of the beadssuch that a bead comprising the unique optical signature may bedistinguished from beads at other locations with different opticalsignatures. Thus the previous work assigned each bioactive agent aunique optical signature such that any microspheres comprising thatbioactive agent are identifiable on the basis of the signature. Theseoptical signatures comprised dyes, usually chromophores or fluorophores,that were entrapped or attached to the beads themselves. Diversity ofoptical signatures utilized different fluorochromes, different ratios ofmixtures of fluorochromes, and different concentrations (intensities) offluorochromes.

Thus, the present invention need not rely solely on the use of opticalproperties to decode the arrays, although in some instances it may.However, as will be appreciated by those in the art, it is possible insome embodiments to utilize optical signatures as an additional codingmethod, in conjunction with the present system. Thus, for example, as ismore fully outlined below, the size of the array may be effectivelyincreased while using a single set of decoding moieties in several ways,one of which is the use in combination with optical signatures onebeads. Thus, for example, using one “set” of decoding molecules, the useof two populations of beads, one with an optical signature and onewithout, allows the effective doubling of the array size. The use ofmultiple optical signatures similarly increases the possible size of thearray.

In a preferred embodiment, each subpopulation of beads comprises aplurality of different IBLs. By using a plurality of different IBLs toencode each bioactive agent, the number of possible unique codes issubstantially increased. That is, by using one unique IBL per bioactiveagent, the size of the array will be the number of unique IBLs (assumingno “reuse” occurs, as outlined below). However, by using a plurality ofdifferent IBLs per bead, n, the size of the array can be increased to2^(n), when the presence or absence of each IBL is used as theindicator. For example, the assignment of 10 IBLs per bead generates a10 bit binary code, where each bit can be designated as “1” (IBL ispresent) or “0” (IBL is absent). A 10 bit binary code has 2¹⁰ possiblevariants However, as is more fully discussed below, the size of thearray may be further increased if another parameter is included such asconcentration or intensity; thus for example, if two differentconcentrations of the IBL are used, then the array size increases as3^(n). Thus, in this embodiment, each individual bioactive agent in thearray is assigned a combination of IBLs, which can be added to the beadsprior to the addition of the bioactive agent, after, or during thesynthesis of the bioactive agent, i.e. simultaneous addition of IBLs andbioactive agent components.

Alternatively, when the bioactive agent is a polymer of differentresidues, i.e. when the bioactive agent is a protein or nucleic acid,the combination of different IBLs can be used to elucidate the sequenceof the protein or nucleic acid.

Thus, for example, using two different IBLs (IBL1 and IBL2), the firstposition of a nucleic acid can be elucidated: for example, adenosine canbe represented by the presence of both IBL1 and IBL2; thymidine can berepresented by the presence of IBL1 but not IBL2, cytosine can berepresented by the presence of IBL2 but not IBL1, and guanosine can berepresented by the absence of both. The second position of the nucleicacid can be done in a similar manner using IBL3 and IBL4; thus, thepresence of IBL1, IBL2, IBL3 and IBL4 gives a sequence of AA; IBL1,IBL2, and IBL3 shows the sequence AT; IBL1, IBL3 and IBL4 gives thesequence TA, etc. The third position utilizes IBL5 and IBL6, etc. Inthis way, the use of 20 different identifiers can yield a unique codefor every possible 10-mer.

The system is similar for proteins but requires a larger number ofdifferent IBLs to identify each position, depending on the alloweddiversity at each position. Thus for example, if every amino acid isallowed at every position, five different IBLs are required for eachposition. However, as outlined above, for example when using randompeptides as the bioactive agents, there may be bias built into thesystem; not all amino acids may be present at all positions, and somepositions may be preset; accordingly, it may be possible to utilize fourdifferent IBLs for each amino acid.

In this way, a sort of “bar code” for each sequence can be constructed;the presence or absence of each distinct IBL will allow theidentification of each bioactive agent.

In addition, the use of different concentrations or densities of IBLsallows a “reuse” of sorts. If, for example, the bead comprising a firstagent has a 1× concentration of IBL, and a second bead comprising asecond agent has a 10× concentration of IBL, using saturatingconcentrations of the corresponding labelled DBL allows the user todistinguish between the two beads.

Once the microspheres comprising the candidate agents and the uniqueIBLs are generated, they are added to the substrate to form an array. Itshould be noted that while most of the methods described herein add thebeads to the substrate prior to the assay, the order of making, usingand decoding the array can vary. For example, the array can be made,decoded, and then the assay done. Alternatively, the array can be made,used in an assay, and then decoded; this may find particular use whenonly a few beads need be decoded. Alternatively, the beads can be addedto the assay mixture, i.e. the sample containing the target analytes,prior to the addition of the beads to the substrate; after addition andassay, the array may be decoded. This is particularly preferred when thesample comprising the beads is agitated or mixed; this can increase theamount of target analyte bound to the beads per unit time, and thus (inthe case of nucleic acid assays) increase the hybridization kinetics.This may find particular use in cases where the concentration of targetanalyte in the sample is low; generally, for low concentrations, longbinding times must be used.

In addition, adding the beads to the assay mixture can allow sorting orselection. For example, a large library of beads may be added to asample, and only those beads that bind the sample may be added to thesubstrate. For example, if the target analyte is fluorescently labeled(either directly (for example by the incorporation of labels intonucleic acid amplification reactions) or indirectly (for example via theuse of sandwich assays)), beads that exhibit fluorescence as a result oftarget analyte binding can be sorted via Fluorescence Activated CellSorting (FACS) and only these beads added to an array and subsequentlydecoded. Similarly, the sorting may be accomplished through affinitytechniques; affinity columns comprising the target analytes can be made,and only those beads which bind are used on the array. Similarly, twobead systems can be used; for example, magnetic beads comprising thetarget analytes can be used to “pull out” those beads that will bind tothe targets, followed by subsequent release of the magnetic beads (forexample via temperature elevation) and addition to an array.

In general, the methods of making the arrays and of decoding the arraysis done to maximize the number of different candidate agents that can beuniquely encoded. The compositions of the invention may be made in avariety of ways. In general, the arrays are made by adding a solution orslurry comprising the beads to a surface containing the sites forassociation of the beads. This may be done in a variety of buffers,including aqueous and organic solvents, and mixtures. The solvent canevaporate, and excess beads removed.

In a preferred embodiment, when non-covalent methods are used toassociate the beads to the array, a novel method of loading the beadsonto the array is used. This method comprises exposing the array to asolution of particles (including microspheres and cells) and thenapplying energy, e.g. agitating or vibrating the mixture. This resultsin an array comprising more tightly associated particles, as theagitation is done with sufficient energy to cause weakly-associatedbeads to fall off (or out, in the case of wells). These sites are thenavailable to bind a different bead. In this way, beads that exhibit ahigh affinity for the sites are selected. Arrays made in this way havetwo main advantages as compared to a more static loading: first of all,a higher percentage of the sites can be filled easily, and secondly, thearrays thus loaded show a substantial decrease in bead loss duringassays. Thus, in a preferred embodiment, these methods are used togenerate arrays that have at least about 50% of the sites filled, withat least about 75% being preferred, and at least about 90% beingparticularly preferred. Similarly, arrays generated in this mannerpreferably lose less than about 20% of the beads during an assay, withless than about 10% being preferred and less than about 5% beingparticularly preferred.

In this embodiment, the substrate comprising the surface with thediscrete sites is immersed into a solution comprising the particles(beads, cells, etc.). The surface may comprise wells, as is describedherein, or other types of sites on a patterned surface such that thereis a differential affinity for the sites. This differential affinityresults in a competitive process, such that particles that willassociate more tightly are selected. Preferably, the entire surface tobe “loaded” with beads is in fluid contact with the solution. Thissolution is generally a slurry ranging from about 10,000:1beads:solution (vol:vol) to 1:1. Generally, the solution can compriseany number of reagents, including aqueous buffers, organic solvents,salts, other reagent components, etc. In addition, the solutionpreferably comprises an excess of beads; that is, there are more beadsthan sites on the array. Preferred embodiments utilize two-fold tobillion-fold excess of beads.

The immersion can mimic the assay conditions; for example, if the arrayis to be “dipped” from above into a microtiter plate comprising samples,this configuration can be repeated for the loading, thus minimizing thebeads that are likely to fall out due to gravity.

Once the surface has been immersed, the substrate, the solution, or bothare subjected to a competitive process, whereby the particles with loweraffinity can be disassociated from the substrate and replaced byparticles exhibiting a higher affinity to the site. This competitiveprocess is done by the introduction of energy, in the form of heat,sonication, stirring or mixing, vibrating or agitating the solution orsubstrate, or both.

A preferred embodiment utilizes agitation or vibration. In general, theamount of manipulation of the substrate is minimized to prevent damageto the array; thus, preferred embodiments utilize the agitation of thesolution rather than the array, although either will work. As will beappreciated by those in the art, this agitation can take on any numberof forms, with a preferred embodiment utilizing microtiter platescomprising bead solutions being agitated using microtiter plate shakers.

The agitation proceeds for a period of time sufficient to load the arrayto a desired fill. Depending on the size and concentration of the beadsand the size of the array, this time may range from about 1 second todays, with from about 1 minute to about 24 hours being preferred.

It should be noted that not all sites of an array may comprise a bead;that is, there may be some sites on the substrate surface which areempty. In addition, there may be some sites that contain more than onebead, although this is not preferred.

In some embodiments, for example when chemical attachment is done, it ispossible to associate the beads in a non-random or ordered way. Forexample, using photoactivatable attachment linkers or photoactivatableadhesives or masks, selected sites on the array may be sequentiallyrendered suitable for attachment, such that defined populations of beadsare laid down.

The arrays of the present invention are constructed such thatinformation about the identity of the candidate agent is built into thearray, such that the random deposition of the beads in the fiber wellscan be “decoded” to allow identification of the candidate agent at allpositions. This may be done in a variety of ways, and either before,during or after the use of the array to detect target molecules.

Thus, after the array is made, it is “decoded” in order to identify thelocation of one or more of the bioactive agents, i.e. each subpopulationof beads, on the substrate surface. FIG. 11 depicts a flow chartexemplifying, but not limiting, the assays that can be performed withthe arrays and hybridization chamber of the invention.

In a preferred embodiment, a selective decoding system is used. In thiscase, only those microspheres exhibiting a change in the optical signalas a result of the binding of a target analyte are decoded. This iscommonly done when the number of “hits”, i.e. the number of sites todecode, is generally low. That is, the array is first scanned underexperimental conditions in the absence of the target analytes. Thesample containing the target analytes is added, and only those locationsexhibiting a change in the optical signal are decoded. For example, thebeads at either the positive or negative signal locations may be eitherselectively tagged or released from the array (for example through theuse of photocleavable linkers), and subsequently sorted or enriched in afluorescence-activated cell sorter (FACS). That is, either all thenegative beads are released, and then the positive beads are eitherreleased or analyzed in situ, or alternatively all the positives arereleased and analyzed. Alternatively, the labels may comprisehalogenated aromatic compounds, and detection of the label is done usingfor example gas chromatography, chemical tags, isotopic tags, or massspectral tags.

As will be appreciated by those in the art, this may also be done insystems where the array is not decoded; i.e. there need not ever be acorrelation of bead composition with location. In this embodiment, thebeads are loaded on the array, and the assay is run. The “positives”,i.e. those beads displaying a change in the optical signal as is morefully outlined below, are then “marked” to distinguish or separate themfrom the “negative” beads. This can be done in several ways, preferablyusing fiber optic arrays. In a preferred embodiment, each bead containsa fluorescent dye. After the assay and the identification of the“positives” or “active beads”, light is shown down either only thepositive fibers or only the negative fibers, generally in the presenceof a light-activated reagent (typically dissolved oxygen). In the formercase, all the active beads are photobleached. Thus, upon non-selectiverelease of all the beads with subsequent sorting, for example using afluorescence activated cell sorter (FACS) machine, the non-fluorescentactive beads can be sorted from the fluorescent negative beads.Alternatively, when light is shown down the negative fibers, all thenegatives are non-fluorescent and the positives are fluorescent, andsorting can proceed. The characterization of the attached bioactiveagent may be done directly, for example using mass spectroscopy.

Alternatively, the identification may occur through the use ofidentifier moieties (“IMs”), which are similar to IBLs but need notnecessarily bind to DBLs. That is, rather than elucidate the structureof the bioactive agent directly, the composition of the IMs may serve asthe identifier. Thus, for example, a specific combination of IMs canserve to code the bead, and be used to identify the agent on the beadupon release from the bead followed by subsequent analysis, for exampleusing a gas chromatograph or mass spectroscope.

Alternatively, rather than having each bead contain a fluorescent dye,each bead comprises a non-fluorescent precursor to a fluorescent dye.For example, using photocleavable protecting groups, such as certainortho-nitrobenzyl groups, on a fluorescent molecule, photoactivation ofthe fluorochrome can be done. After the assay, light is shown down againeither the “positive” or the “negative” fibers, to distinguish thesepopulations. The illuminated precursors are then chemically converted toa fluorescent dye. All the beads are then released from the array, withsorting, to form populations of fluorescent and non-fluorescent beads(either the positives and the negatives or vice versa).

In an alternate preferred embodiment, the sites of association of thebeads (for example the wells) include a photopolymerizable reagent, orthe photopolymerizable agent is added to the assembled array. After thetest assay is run, light is shown down again either the “positive” orthe “negative” fibers, to distinguish these populations. As a result ofthe irradiation, either all the positives or all the negatives arepolymerized and trapped or bound to the sites, while the otherpopulation of beads can be released from the array.

In a preferred embodiment, the location of every bioactive agent isdetermined using decoder binding ligands (DBLs). As outlined above, DBLsare binding ligands that will either bind to identifier binding ligands,if present, or to the bioactive agents themselves, preferably when thebioactive agent is a nucleic acid or protein.

In a preferred embodiment, as outlined above, the DBL binds to the IBL.

In a preferred embodiment, the bioactive agents are single-strandednucleic acids and the DBL is a substantially complementarysingle-stranded nucleic acid that binds (hybridizes) to the bioactiveagent, termed a decoder probe herein. A decoder probe that issubstantially complementary to each candidate probe is made and used todecode the array. In this embodiment, the candidate probes and thedecoder probes should be of sufficient length (and the decoding step rununder suitable conditions) to allow specificity; i.e. each candidateprobe binds to its corresponding decoder probe with sufficientspecificity to allow the distinction of each candidate probe.

In a preferred embodiment, the DBLs are either directly or indirectlylabeled. By “labeled” herein is meant that a compound has at least oneelement, isotope or chemical compound attached to enable the detectionof the compound. In general, labels fall into three classes: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic,electrical, thermal; and c) colored or luminescent dyes; although labelsinclude enzymes and particles such as magnetic particles as well.Preferred labels include luminescent labels. In a preferred embodiment,the DBL is directly labeled, that is, the DBL comprises a label. In analternate embodiment, the DBL is indirectly labeled; that is, a labelingbinding ligand (LBL) that will bind to the DBL is used. In thisembodiment, the labeling binding ligand-DBL pair can be as describedabove for IBL-DBL pairs. Suitable labels include, but are not limitedto, fluorescent lanthanide complexes, including those of Europium andTerbium, fluorescein, rhodamine, tetramethylrhodamine, eosin,erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, FITC, PE, cy3, cy5and others described in the 6th Edition of the Molecular Probes Handbookby Richard P. Haugland, hereby expressly incorporated by reference.

In one embodiment, the label is a molecule whose color or luminescenceproperties change in the presence of the IBL, due to a change in thelocal environment. For example, the label may be: (1) a fluorescent pHindicator whose emission intensity changes with pH; (2) a fluorescention indicator, whose emission properties change with ion concentration;or (3) a fluorescent molecule such as an ethidium salt whosefluorescence intensity increases in hydrophobic environments.

Accordingly, the identification of the location of the individual beads(or subpopulations of beads) is done using one or more decoding stepscomprising a binding between the labeled DBL and either the IBL or thebioactive agent (i.e. a hybridization between the candidate probe andthe decoder probe when the bioactive agent is a nucleic acid). Afterdecoding, the DBLs can be removed and the array can be used; however, insome circumstances, for example when the DBL binds to an IBL and not tothe bioactive agent, the removal of the DBL is not required (although itmay be desirable in some circumstances). In addition, as outlinedherein, decoding may be done either before the array is used in anassay, during the assay, or after the assay.

In one embodiment, a single decoding step is done. In this embodiment,each DBL is labeled with a unique label, such that the number of uniquelabels is equal to or greater than the number of bioactive agents(although in some cases, “reuse” of the unique labels can be done, asdescribed herein; similarly, minor variants of candidate probes canshare the same decoder, if the variants are encoded in anotherdimension, i.e. in the bead size or label). For each bioactive agent orIBL, a DBL is made that will specifically bind to it and contains aunique label, for example one or more fluorochromes. Thus, the identityof each DBL, both its composition (i.e. its sequence when it is anucleic acid) and its label, is known. Then, by adding the DBLs to thearray containing the bioactive agents under conditions which allow theformation of complexes (termed hybridization complexes when thecomponents are nucleic acids) between the DBLs and either the bioactiveagents or the IBLs, the location of each DBL can be elucidated. Thisallows the identification of the location of each bioactive agent; therandom array has been decoded. The DBLs can then be removed, ifnecessary, and the target sample applied.

In a preferred embodiment, the number of unique labels is less than thenumber of unique bioactive agents, and thus a sequential series ofdecoding steps are used. To facilitate the discussion, this embodimentis explained for nucleic acids, although other types of bioactive agentsand DBLs are useful as well. In this embodiment, decoder probes aredivided into n sets for decoding. The number of sets corresponds to thenumber of unique tags. Each decoder probe is labeled in n separatereactions with n distinct tags. All the decoder probes share the same ntags. Each pool of decoders contains only one of the n tag versions ofeach decoder, and no two decoder probes have the same sequence of tagsacross all the pools. The number of pools required for this to be trueis determined by the number of decoder probes and the n. Hybridizationof each pool to the array generates a signal at every address comprisingan IBL. The sequential hybridization of each pool in turn will generatea unique, sequence-specific code for each candidate probe. Thisidentifies the candidate probe at each address in the array. Forexample, if four tags are used, then 4×n sequential hybridizations canideally distinguish 4^(n) sequences, although in some cases more stepsmay be required. After the hybridization of each pool, the hybrids aredenatured and the decoder probes removed, so that the probes arerendered single-stranded for the next hybridization (although it is alsopossible to hybridize limiting amounts of target so that the availableprobe is not saturated. Sequential hybridizations can be carried out andanalyzed by subtracting preexisting signal from the previoushybridization).

As will be appreciated by one of ordinary skill in the art,hybridization or incubation times vary. Generally, hybridization orincubation times last from seconds to minutes or up to hours or days ormore. When the hybridization chamber as described herein is utilized,hybridization or incubation times can be increased relative toincubation times without the hybridization chamber.

An example is illustrative. Assuming an array of 16 probe nucleic acids(numbers 1-16), and four unique tags (four different fluors, forexample; labels A-D). Decoder probes 1-16 are made that correspond tothe probes on the beads. The first step is to label decoder probes 14with tag A, decoder probes 5-8 with tag B, decoder probes 9-12 with tagC, and decoder probes 13-16 with tag D. The probes are mixed and thepool is contacted with the array containing the beads with the attachedcandidate probes. The location of each tag (and thus each decoder andcandidate probe pair) is then determined. The first set of decoderprobes are then removed. A second set is added, but this time, decoderprobes 1, 5, 9 and 13 are labeled with tag A, decoder probes 2, 6, 10and 14 are labeled with tag B, decoder probes 3, 7, 11 and 15 arelabeled with tag C, and decoder probes 4, 8, 12 and 16 are labeled withtag D. Thus, those beads that contained tag A in both decoding stepscontain candidate probe 1; tag A in the first decoding step and tag B inthe second decoding step contain candidate probe 2; tag A in the firstdecoding step and tag C in the second step contain candidate probe 3;etc. As will be appreciated by those in the art, the decoder probes canbe made in any order and added in Any order.

In one embodiment, the decoder probes are labeled in situ; that is, theyneed not be labeled prior to the decoding reaction. In this embodiment,the incoming decoder probe is shorter than the candidate probe, creatinga 5′ “overhang” on the decoding probe. The addition of labeled ddNTPs(each labeled with a unique tag) and a polymerase will allow theaddition of the tags in a sequence specific manner, thus creating asequence-specific pattern of signals. Similarly, other modifications canbe done, including ligation, etc.

In addition, since the size of the array will be set by the number ofunique decoding binding ligands, it is possible to “reuse” a set ofunique DBLs to allow for a greater number of test sites. This may bedone in several ways; for example, by using some subpopulations thatcomprise optical signatures. Similarly, the use of a positional codingscheme within an array; different sub-bundles may reuse the set of DBLs.Similarly, one embodiment utilizes bead size as a coding modality, thusallowing the reuse of the set of unique DBLs for each bead size.Alternatively, sequential partial loading of arrays with beads can alsoallow the reuse of DBLs. Furthermore, “code sharing” can occur as well.

In a preferred embodiment, the DBLs may be reused by having somesubpopulations of beads comprise optical signatures. In a preferredembodiment, the optical signature is generally a mixture of reporterdyes, preferably fluorescent. By varying both the composition of themixture (i.e. the ratio of one dye to another) and the concentration ofthe dye (leading to differences in signal intensity), matrices of uniqueoptical signatures may be generated. This may be done by covalentlyattaching the dyes to the surface of the beads, or alternatively, byentrapping the dye within the bead. The dyes may be chromophores orphosphors but are preferably fluorescent dyes, which due to their strongsignals provide a good signal-to-noise ratio for decoding. Suitable dyesfor use in the invention include those listed for labeling DBLs, above.

In a preferred embodiment, the encoding can be accomplished in a ratioof at least two dyes, although more encoding dimensions may be added inthe size of the beads, for example. In addition, the labels aredistinguishable from one another; thus two different labels may comprisedifferent molecules (i.e. two different fluors) or, alternatively, onelabel at two different concentrations or intensity.

In a preferred embodiment, the dyes are covalently attached to thesurface of the beads. This may be done as is generally outlined for theattachment of the bioactive agents, using functional groups on thesurface of the beads. As will be appreciated by those in the art, theseattachments are done to minimize the effect on the dye.

In a preferred embodiment, the dyes are non-covalently associated withthe beads, generally by entrapping the dyes in the pores of the beads.

Additionally, encoding in the ratios of the two or more dyes, ratherthan single dye concentrations, is preferred since it providesinsensitivity to the intensity of light used to interrogate the reporterdye's signature and detector sensitivity.

In a preferred embodiment, a spatial or positional coding system isdone. In this embodiment, there are sub-bundles or subarrays (i.e.portions of the total array) that are utilized. By analogy with thetelephone system, each subarray is an “area code”, that can have thesame labels (i.e. telephone numbers) of other subarrays, that areseparated by virtue of the location of the subarray. Thus, for example,the same unique labels can be reused from bundle to bundle. Thus, theuse of 50 unique labels in combination with 100 different subarrays canform an array of 5000 different bioactive agents. In this embodiment, itbecomes important to be able to identify one bundle from another; ingeneral, this is done either manually or through the use of markerbeads; these can be beads containing unique tags for each subarray, orthe use of the same marker bead in differing amounts, or the use of twoor more marker beads in different ratios.

In alternative embodiments, additional encoding parameters can be added,such as microsphere size. For example, the use of different size beadsmay also allow the reuse of sets of DBLs; that is, it is possible to usemicrospheres of different sizes to expand the encoding dimensions of themicrospheres. Optical fiber arrays can be fabricated containing pixelswith different fiber diameters or cross-sections; alternatively, two ormore fiber optic bundles, each with different cross-sections of theindividual fibers, can be added together to form a larger bundle; or,fiber optic bundles with fiber of the same size cross-sections can beused, but just with different sized beads. With different diameters, thelargest wells can be filled with the largest microspheres and thenmoving onto progressively smaller microspheres in the smaller wellsuntil all size wells are then filled. In this manner, the same dye ratiocould be used to encode microspheres of different sizes therebyexpanding the number of different oligonucleotide sequences or chemicalfunctionalities present in the array. Although outlined for fiber opticsubstrates, this as well as the other methods outlined herein can beused with other substrates and with other attachment modalities as well.

In a preferred embodiment, the coding and decoding is accomplished bysequential loading of the microspheres into the array. As outlined abovefor spatial coding, in this embodiment, the optical signatures can be“reused”. In this embodiment, the library of microspheres eachcomprising a different bioactive agent (or the subpopulations eachcomprise a different bioactive agent), is divided into a plurality ofsublibraries; for example, depending on the size of the desired arrayand the number of unique tags, 10 sublibraries each comprising roughly10% of the total library may be made, with each sublibrary comprisingroughly the same unique tags. Then, the first sublibrary is added to thefiber optic bundle comprising the wells, and the location of eachbioactive agent is determined, generally through the use of DBLs. Thesecond sublibrary is then added, and the location of each bioactiveagent is again determined. The signal in this case will comprise thesignal from the “first” DBL and the “second” DBL; by comparing the twomatrices the location of each bead in each sublibrary can be determined.Similarly, adding the third, fourth, etc. sublibraries sequentially willallow the array to be filled.

In a preferred embodiment, codes can be “shared” in several ways. In afirst embodiment, a single code (i.e. IBL/DBL pair) can be assigned totwo or more agents if the target analytes different sufficiently intheir binding strengths. For example, two nucleic acid probes used in anmRNA quantitation assay can share the same code if the ranges of theirhybridization signal intensities do not overlap. This can occur, forexample, when one of the target sequences is always present at a muchhigher concentration than the other. Alternatively, the two targetsequences might always be present at a similar concentration, but differin hybridization efficiency.

Alternatively, a single code can be assigned to multiple agents if theagents are functionally equivalent. For example, if a set ofoligonucleotide probes are designed with the common purpose of detectingthe presence of a particular gene, then the probes are functionallyequivalent, even though they may differ in sequence. Similarly, ifclasses or “families” of analytes are desired, all probes for differentmembers of a class such as kinases or G-protein coupled receptors couldshare a code. Similarly, an array of this type could be used to detecthomologs of known genes. In this embodiment, each gene is represented bya heterologous set of probes, hybridizing to different regions of thegene (and therefore differing in sequence). The set of probes share acommon code. If a homolog is present, it might hybridize to some but notall of the probes. The level of homology might be indicated by thefraction of probes hybridizing, as well as the average hybridizationintensity. Similarly, multiple antibodies to the same protein could allshare the same code.

In a preferred embodiment, decoding of self-assembled random arrays isdone on the bases of pH titration. In this embodiment, in addition tobioactive agents, the beads comprise optical signatures, wherein theoptical signatures are generated by the use of pH-responsive dyes(sometimes referred to herein as “pH dyes”) such as fluorophores. Thisembodiment is similar to that outlined in PCT US98/05025 and U.S. Ser.No. 09/151,877, both of which are expressly incorporated by reference,except that the dyes used in the present invention exhibits changes influorescence intensity (or other properties) when the solution pH isadjusted from below the pKa to above the pKa (or vice versa). In apreferred embodiment, a set of pH dyes is used, each with a differentpKa, preferably separated by at least 0.5 pH units. Preferredembodiments utilize a pH dye set of pKa's of 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11,and 11.5. Each bead can contain any subset of the pH dyes, and in thisway a unique code for the bioactive agent is generated. Thus, thedecoding of an array is achieved by titrating the array from pH 1 to pH13, and measuring the fluorescence signal from each bead as a functionof solution pH.

In a preferred embodiment, there are additional ways to increase thenumber of unique or distinct tags. That is, the use of distinctattributes on each bead can be used to increase the number of codes. Inaddition, sequential decoding allows a reuse of codes in new ways. Theseattributes are independent of each other, thus allowing the number ofcodes to grow exponentially as a function of the number of decodingsteps and the number of attributes (e.g. distinct codes). However, byincreasing the amount of decoding information obtained in a singledecoding step, the number of decoding steps is markedly reduced.Alternatively, the number of distinct codes is markedly increased. Byincreasing the number of attributes per decoding step, fewer decodingsteps are required for a given number of codes. Thus, in a preferredembodiment, a variety of methods are used to generate a number of codesfor use in the process of decoding the arrays, while minimizing thenecessary decoding steps. For example, a variety of different codingstrategies can be combined: thus, different “colors”, combinations ofcolors (“hues”), different intensities of colors or hues or both, etc.can all be combined.

In a preferred embodiment DBLs rely on attaching or embedding aquantitative or discrete set of physical attributes to the bead, i.e.labeling the bead. Preferred physical attributes of a bead include butare not limited to: surface “smoothness” or “roughness”, color(Fluorescent and otherwise), color intensity, size, detectable chemicalmoieties, chemical reactivity, magnetization, pH sensitivity, energytransfer efficiency between dyes present, hydrophobicity,hydrophilicity, absorptivity, charge, pH sensitivity, etc.

A bead decoding scheme includes assigning/imbuing a single quantifiableattribute to each bead type wherein each bead type differs in thequantifiable value of that attribute. For instance, one can attach agiven number of fluorophores to a bead and quantitate the number ofattached fluorophores in the decoding process; however, in practice,attaching a “given amount” of an attribute to a bead and accuratelymeasuring the attribute may be problematic. In general, the goal is toreduce the coefficient of variation (CV). By coefficient of variation ismeant the variability in labeling a bead in successive labelings. ThisCV can be determined by labeling beads with a defined given number oflabel (fluorophore, for example) in multiple tests and measuring theresulting signal emitted by the bead. A large CV limits the number ofuseable and resolvable “levels” for any given attribute.

A more robust decoding scheme employs ratiometric rather than absolutemeasurements for segmenting a quantitative attribute into codes. Byratiometric decoding is meant labeling a bead with a ratio of labels(i.e. 1:10, 1:1, and 10:1). In theory any number of ratios can be usedso long as the difference in signals between the ratios is detectable.This process produced smaller CVs and allowing more attributesegmentation within a given dynamic range. Thus, in a preferredembodiment, the use of ratiometric decoding reduces the coefficient ofvariability.

In addition, as will be appreciated by those in the art, ratiometricdecoding can be accomplished in a different way. In this embodiment,rather than add a given number of DBLs with a first dye (or dyecombination) intensity in the first decoding reaction and a secondnumber with a second dye intensity in the sequential second decodingreaction, this ratiometric analysis may be done by using a ratio oflabelled:unlabelled DBLs. That is, given a set saturating concentrationof decoding beads, for example 100,000 DBLs/reaction, the firstintensity decoding step may be done by adding 100,000 labelled DBLs andthe second step can be done by adding 10,000 labelled DBLs and 90,000unlabeled DBLs. Equilibrium dictates that the second step will give onetenth the signal intensity.

Because of the spread in values of a quantitatively measured attributevalue, the number of distinct codes is practically limited to less thana dozen or so codes. However, by serially “painting” (i.e. temporarilyattaching an attribute level to a bead) and “stripping” (removing theattribute level) a bead with different attribute values, the number ofpossible codes grows exponentially with the number of serial stages inthe decoding process.

An example is illustrative. For instance, 9 different bead types andthree distinguishable attribute distributions (Table 1). “Painting”(labeling) the beads with different attribute values in acombinatorially distinct pattern in the two different stages, generatesa unique code for each bead type, i.e. nine distinct codes aregenerated. Thus, in a preferred embodiment beads are labeled withdifferent attributes in a combinatorially distinct pattern in aplurality of stages. This generates unique codes for each bead type.Examples of different attributes are described above. Labeling of beadswith different attributes is performed by methods known in the art.

TABLE 1 Serial decode generates unique codes using a small number ofattribute levels. stage 1 stage 2 Bead attribute attribute Type valuevalue Code 1 L L (L, L) 2 L M (L, M) 3 L H (L, H) 4 M L (M, L) 5 M M (M,M) 6 M H (M, H) 7 H L (H, L) 8 H M (H, M) 9 H H (H, H) Number of uniquecodes = Number of attributes{circumflex over ( )}Number of stages

Fluorescent colors are a particularly convenient attribute to use in adecoding scheme. Fluorescent colors can be attached to any agent thatrecognizes an IBL to form a labeled DBL. The discussion is directed tooligonucleotides (including nucleic acid analogs) as the DBLs. Afluorescently labeled oligonucleotide is a particularly useful DBL sinceit can specifically and reversibly “paint” (label) any desired subset ofbeads with a particular color simply by the process of hybridization anddehybridization (i.e. to the DBL with a complementary sequence).Moreover, fluorescence is easily imaged and quantitated using standardoptical hardware and software. In order to “paint” a given bead typewith a particular color, the bead type must be labeled with a uniquehybridizable DNA sequence (IBL) and the decoding solution must containthe color-labeled complement of that sequence.

One consideration in implementing a decoding scheme is to minimize thenumber of images collected. In a color-based scheme, the number ofimages collected is the product of the number of colors and the numberof stages. The number of images can be reduced by “painting” a bead withmultiple colors for each given stage. By assigning multiple colors to abead, the number of effective codes is increased. As an example, in a 24bit three color scheme (e.g. red, green, blue) coloring process used bycomputers, a total of 256*256*256=16.7 million different “hues” can begenerated from just three colors (red, green, blue).

Thus, in a preferred embodiment DBLs are labeled with a combination ofcolored fluorophores. As such, this method finds use in increasing thenumber of available codes for labeling DBLs using only a handful ofdifferent dyes (colors). Increasing the number of codes available ateach decoding step will greatly decrease the number of decoding stepsrequired in a given decoding process.

In one embodiment a population of oligonucleotides encoding a single DBLis labeled with a defined ratio of colors such that each bead to whichthe DBL binds is identified based on a characteristic “hue” formulatedfrom the combination of the colored fluorophores. In a preferredembodiment two distinct colors are used. In a preferred embodiment,three or more distinct dyes (colors) are available for use. In thisinstance the number of differentiable codes generated by labeling apopulation of oligonucleotides encoding a single DBL with any givencolor is three. However by allowing combinations of colors and colorlevels in the labeling, many more codes are generated.

For decoding by hybridization, a preferred number of distinguishablecolor shades is from 2 to 2000; a more preferred number ofdistinguishable color shades is from 2 to 200 and a most preferrednumber of distinguishable color shades is from 2 to 20. Utilizing threedifferent color shades (intensities) and three colors, the number ofdifferent hues will be 34=81. Combining a hue with sequential decodingallows a virtually limitless number of codes to be generated.

As previously described, the DBL can be any agent that binds to the IBL.In a preferred embodiment, a single DBL is labeled with a pre-determinedratio of colors. This ratio is varied for each DBL thus allowing for aunique “hue” for each DBL labeled as such. Following treatment of thebeads with the DBL, the bead is analyzed to determine the “hue”associated with each bead, thereby identifying the bead with itsassociated bioactive agent.

For instance, with four primary colors and two intensity levels (coloris present or absent), fifteen different hues/stage are possible. Iffour dyes and three different intensity levels are used (absent,half-present, fully present), then 73 different hues/stage are possible.In this case, acquisition of only 4 color images is sufficient to obtaininformation on 73 different coding hues.

In a preferred embodiment, the present invention provides arraycompositions comprising a first substrate with a surface comprisingdiscrete sites. Preferred embodiments utilize a population ofmicrospheres distributed on the sites, and the population comprises atleast a first and a second subpopulation. Each subpopulation comprises abioactive agent, and, in addition, at least one optical dye with a givenpKa. The pKas of the different optical dyes are different.

In a preferred embodiment, when for example the array comprises clonednucleic acids, there are several methods that can be used to decode thearrays. In a preferred embodiment, when some sequence information aboutthe cloned nucleic acids is known, specific decoding probes can be madeas is generally outlined herein.

In a preferred embodiment, “random” decoding probes can be made. Bysequential hybridizations or the use of multiple labels, as is outlinedabove, a unique hybridization pattern can be generated for each sensorelement. This allows all the beads representing a given clone to beidentified as belonging to the same group. In general, this is done byusing random or partially degenerate decoding probes, that bind in asequence-dependent but not highly sequence-specific manner. The processcan be repeated a number of times, each time using a different labelingentity, to generate a different pattern of signals based onquasi-specific interactions. In this way, a unique optical signature iseventually built up for each sensor element. By applying patternrecognition or clustering algorithms to the optical signatures, thebeads can be grouped into sets that share the same signature (i.e. carrythe same probes).

In order to identify the actual sequence of the clone itself, additionalprocedures are required; for example, direct sequencing can be done. Byusing an ordered array containing the clones, such as a spotted cDNAarray, a “key” can be generated that links a hybridization pattern to aspecific clone whose position in the set is known. In this way the clonecan be recovered and further characterized.

Alternatively, clone arrays can be decoded using binary decoding withvector tags. For example, partially randomized oligos are cloned into anucleic acid vector (e.g. plasmid, phage, etc.). Each oligonucleotidesequence consists of a subset of a limited set of sequences. Forexample, if the limits set comprises 10 sequences, each oligonucleotidemay have some subset (or all of the 10) sequences. Thus each of the 10sequences can be present or absent in the oligonucleotide. Therefore,there are 210 or 1,024 possible combinations. The sequences may overlap,and minor variants can also be represented (e.g. A, C, T and Gsubstitutions) to increase the number of possible combinations. Anucleic acid library is cloned into a vector containing the random codesequences. Alternatively, other methods such as PCR can be used to addthe tags. In this way it is possible to use a small number of oligodecoding probes to decode an array of clones.

In a preferred embodiment, discriminant analysis and cluster algorithmsand computer apparatus are used to analyze the decoding data from thearrays of the invention. The potentially large number of codes utilizedin the invention, coupled with the use of different intensities and“hues” of fluorophores in multi-step decoding processes requires goodclassification of the data. The data, particularly intensity data, isacquired in a multi-step process during which beads are reversiblylabeled (for example by hybridizing dye-labeled complementary decodingoligonucleotides to the IBL probes on the beads, or the formation ofbinding ligand pairs for non-nucleic acid IBL-DBL pairs) with differentcolors or mixtures of colors (“hues”) at each stage. The challenge is toaccurately classify a bead as to which color with which it was paintedat each step. The more closely related the labels are to one another (asdetermined by the optical imaging system), the more difficult theclassification.

The proximity of the dyes as seen by the imaging system is determined bythe spectral properties of the decoding dyes and the spectral channelseparation of the imaging system. Better color separation is achieved byemploying fluorescent dyes with narrow emission spectra, and byemploying an optical system with narrow band pass excitation andemission filters which are designed to excite the dye “on peak” andmeasure its emission “on peak”. The process of optically imaging thedyes on the beads is similar to the human vision process in which ourbrain sees color by measuring the ratio of excitation in the threedifferent cone types within our eye. However, with an optical imagingsystem, the number of practical color channels is much greater than thethree present in the human eye. CCD based imaging systems can “see”color from 350 nm up to 850 nm whereas the cones in the eye are tuned tothe visible spectrum from 500-600 nm.

The problem of decoding bead arrays is essentially a discriminantanalysis classification problem. Thus, in a preferred embodiment, ananalysis of variance in hyperspectral alpha space is performed on aknown set of bead colors or hues. The center of the bead clusters inalpha space are termed the centroids of the clusters, and the scatter ofthe points within a cluster determines the spread of the cluster. Arobust classification scheme requires that the distance between thecentroids of the different bead classes (hues) is much greater than thespread of any cluster class. Moreover, the location of the centroidsshould remain invariant from fiber to fiber and from experiment toexperiment. Thus, in a preferred embodiment, a hue “zone” is defined asa region in alpha space surrounding the hue centroid and extending outto the spread radius of the cluster. Given a reference set of huecentroids and spread radii, as determined empirically, theclassification of a new set of data can be accomplished by askingwhether a given bead point falls closest to or within the “zone” of ahue cluster. This is accomplished by calculating the Mahalanobisdistance (in this case, it is simply a Euclidean distance metric) of thebead point from the centroids of the different hue classes. For the datashown in FIG. 3, the location of the centroids and their distances fromone another are indicated in Table 2.

TABLE 2 Centroid position Distance between centroids dye/channel Bod-Bod- Bod- Bod- Blue Green Yellow Red 493 R6G 564 TXR Bod-493 0.63 0.220.11 0.03 0.00 Bod-R6G 0.03 0.51 0.37 0.09 0.72 0.00 Bod-564 0.06 0.040.57 0.32 0.81 0.55 0.00 Bod-TXR 0.09 0.05 0.04 0.82 0.99 0.93 0.73 0.00

For classifying the different beads into a particular hue class, aEuclidean distance cutoff of 0.3 was chosen. The closest two centroids,the Bod-R6G and Bod-564 (dist=0.55), have a slight overlap in theirdecoding zones when using a Euclidean or Mahalanobis distance of 0.3. Animprovement in classification can be achieved by decreasing thisdistance, and by weighting the different coordinate axes appropriately.

Accordingly, the present invention provides computer methods foranalyzing and classifying the color of a bead. The classification of thecolor of the bead is done by viewing the bead in hyperspectral “alpha”space (a₁=I₁/SI_(I), a₂=I₂/SI_(i), a₃=I₃/SI_(i), etc.) in which eachcoordinate axis represents the fraction of the bead intensity within agiven imaging channel. For instance, if four imaging channels are usedto image the beads, the color or hue of a bead can be represented by apoint in 3-D alpha space (the fourth dimension is not necessary sinceSa_(i)=1). Given a set of different primary dyes by which to label thebeads, the number of hues that can be generated from these dyes isunlimited since the dyes can be combined in varying ratios and invarying combinatorial patterns. The number of practical hues isexperimentally determined by the separation of the different hueclusters in hyperspectral alpha space.

FIG. 3 shows a hyperspectral alpha plot of beads labeled with fourdifferent hues imaged in four separate imaging channels. Note that thebeads form four distinct clusters. The fact that these four clusters arewell separated allows a robust decode classification scheme to beimplemented.

In a preferred embodiment, a quality control analysis of the decodingprocess is done. This is achieved by performing a cluster analysis ofalpha space for each decoding stage. The number of clusters determinedwill be fixed by the expected number of hues. The positions of thecluster centroids will be monitored and any deviations from the expectedposition will be noted.

Thus the invention provides an apparatus for decoding the arrays of theinvention. In addition to the compositions outlined herein, theapparatus includes a central processing unit which communicates with amemory and a set of input/output devices (e.g., keyboard, mouse,monitor, printer, etc.) through a bus. The general interaction between acentral processing unit, a memory, input/output devices, and a bus isknown in the art. One aspect of the present invention is directed towardthe hyperspectral “alpha” space classification system stored in thememory.

The classification system program includes a data acquisition modulethat receives data from the optical reader or confocal microscope (orother imaging system). In general, the classification program alsoincludes an analysis module, that can analyze the variance inhyperspectral alpha space, calculate the centroids of the clusters,calculate the scatter of the cluster (the spread) and define the huezone and distance cutoff. In general, the analysis module will furtherdetermine whether a data point falls within the hue zone by calculatingthe Mahalanobis distance.

Finally, the analysis module will analyze the different sequentialdecoding information to finally assign a bioactive agent to a beadlocation.

In this way, sequential decoding steps are run, with each step utilizingthe discriminant analysis calculations to assign each bead in the arrayto a hue cluster at each step. The buildup of the sequential decodinginformation allows the correlation of the location of a bead and thechemistry contained on it.

Once made, the compositions of the invention find use in a number ofapplications. In a preferred embodiment, the compositions are used toprobe a sample solution for the presence or absence of a target analyte,including the quantification of the amount of target analyte present. By“target analyte” or “analyte” or grammatical equivalents herein is meantany atom, molecule, ion, molecular ion, compound or particle to beeither detected or evaluated for binding partners. As will beappreciated by those in the art, a large number of analytes may be usedin the present invention; basically, any target analyte can be usedwhich binds a bioactive agent or for which a binding partner (i.e. drugcandidate) is sought.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. When detection of a target analyte is done, suitabletarget analytes include, but are not limited to, an environmentalpollutant (including pesticides, insecticides, toxins, etc.); a chemical(including solvents, polymers, organic materials, etc.); therapeuticmolecules (including therapeutic and abused drugs, antibiotics, etc.);biomolecules (including hormones, cytokines, proteins, nucleic acids,lipids, carbohydrates, cellular membrane antigens and receptors (neural,hormonal, nutrient, and cell surface receptors) or their ligands, etc);whole cells (including procaryotic (such as pathogenic bacteria) andeukaryotic cells, including mammalian tumor cells); viruses (includingretroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); andspores; etc. Particularly preferred analytes are nucleic acids andproteins.

In a preferred embodiment, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous target analytes that may be detected or evaluated forbinding partners using the present invention. Suitable protein targetanalytes include, but are not limited to, (1) immunoglobulins; (2)enzymes (and other proteins); (3) hormones and cytokines (many of whichserve as ligands for cellular receptors); and (4) other proteins.

In a preferred embodiment, the target analyte is a nucleic acid. Theseassays find use in a wide variety of applications, as is generallyoutlined in U.S. Ser. Nos. 60/160,027; 60/161,148; 09/425,633; and60/160,917, all of which are expressly incorporated herein by reference.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, cytochrome p450s or anyof the others well known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, chlamydia and othersexually transmitted diseases, may also be detected.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid, and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E.coli, and Legionnaire's disease bacteria. Similarly, bioremediationstrategies may be evaluated using the compositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

The present invention also finds use as a methodology for the detectionof mutations or mismatches in target nucleic acid sequences. Forexample, recent focus has been on the analysis of the relationshipbetween genetic variation and phenotype by making use of polymorphic DNAmarkers. Previous work utilized short tandem repeats (STRs) aspolymorphic positional markers; however, recent focus is on the use ofsingle nucleotide polymorphisms (SNPs), which occur at an averagefrequency of more than 1 per kilobase in human genomic DNA. Some SNPs,particularly those in and around coding sequences, are likely to be thedirect cause of therapeutically relevant phenotypic variants. There area number of well known polymorphisms that cause clinically importantphenotypes; for example, the apoE2/3/4 variants are associated withdifferent relative risk of Alzheimer's and other diseases (see Cordor etal., Science 261 (1993). Multiplex PCR amplification of SNP loci withsubsequent hybridization to oligonucleotide arrays has been shown to bean accurate and reliable method of simultaneously genotyping at leasthundreds of SNPs; see Wang et al., Science, 280:1077 (1998); see alsoSchafer et al., Nature Biotechnology 16:33-39 (1998). The compositionsof the present invention may easily be substituted for the arrays of theprior art; in particular, single base extension (SBE) and pyrosequencingtechniques are particularly useful with the compositions of theinvention.

In a preferred embodiment, the compositions of the invention are used toscreen bioactive agents to find an agent that will bind, and preferablymodify the function of, a target molecule. As above, a wide variety ofdifferent assay formats may be run, as will be appreciated by those inthe art. Generally, the target analyte for which a binding partner isdesired is labeled; binding of the target analyte by the bioactive agentresults in the recruitment of the label to the bead, with subsequentdetection.

In a preferred embodiment, the binding of the bioactive agent and thetarget analyte is specific; that is, the bioactive agent specificallybinds to the target analyte. By “specifically bind” herein is meant thatthe agent binds the analyte, with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. However, as will be appreciated by those in the art,it will be possible to detect analytes using binding which is not highlyspecific; for example, the systems may use different binding ligands,for example an array of different ligands, and detection of anyparticular analyte is via its “signature” of binding to a panel ofbinding ligands, similar to the manner in which “electronic noses” work.This finds particular utility in the detection of chemical analytes. Thebinding should be sufficient to remain bound under the conditions of theassay, including wash steps to remove non-specific binding, although insome embodiments, wash steps are not desired; i.e. for detecting lowaffinity binding partners. In some embodiments, for example in thedetection of certain biomolecules, the dissociation constants of theanalyte to the binding ligand will be less than about 10⁻⁴-10⁻⁶ M⁻¹,with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ being preferred and less thanabout 10⁻⁷-10⁻⁹ M⁻¹ being particularly preferred.

Generally, a sample containing a target analyte (whether for detectionof the target analyte or screening for binding partners of the targetanalyte) is added to the array, under conditions suitable for binding ofthe target analyte to at least one of the bioactive agents, i.e.generally physiological conditions. The presence or absence of thetarget analyte is then detected. As will be appreciated by those in theart, this may be done in a variety of ways, generally through the use ofa change in an optical signal. This change can occur via many differentmechanisms. A few examples include the binding of a dye-tagged analyteto the bead, the production of a dye species on or near the beads, thedestruction of an existing dye species, a change in the opticalsignature upon analyte interaction with dye on bead, or any otheroptical interrogatable event.

In a preferred embodiment, the change in optical signal occurs as aresult of the binding of a target analyte that is labeled, eitherdirectly or indirectly, with a detectable label, preferably an opticallabel such as a fluorochrome. Thus, for example, when a proteinaceoustarget analyte is used, it may be either directly labeled with a fluor,or indirectly, for example through the use of a labeled antibody.Similarly, nucleic acids are easily labeled with fluorochromes, forexample during PCR amplification as is known in the art. Alternatively,upon binding of the target sequences, a hybridization indicator may beused as the label. Hybridization indicators preferentially associatewith double stranded nucleic acid, usually reversibly. Hybridizationindicators include intercalators and minor and/or major groove bindingmoieties. In a preferred embodiment, intercalators may be used; sinceintercalation generally only occurs in the presence of double strandednucleic acid, only in the presence of target hybridization will thelabel light up. Thus, upon binding of the target analyte to a bioactiveagent, there is a new optical signal generated at that site, which thenmay be detected.

Alternatively, in some cases, as discussed above, the target analytesuch as an enzyme generates a species that is either directly orindirectly optical detectable.

Furthermore, in some embodiments, a change in the optical signature maybe the basis of the optical signal. For example, the interaction of somechemical target analytes with some fluorescent dyes on the beads mayalter the optical signature, thus generating a different optical signal.

As will be appreciated by those in the art, in some embodiments, thepresence or absence of the target analyte may be done using changes inother optical or non-optical signals, including, but not limited to,surface enhanced Raman spectroscopy, surface plasmon resonance,radioactivity, etc.

The assays may be run under a variety of experimental conditions, aswill be appreciated by those in the art. A variety of other reagents maybe included in the screening assays. These include reagents like salts,neutral proteins, e.g. albumin, detergents, etc which may be used tofacilitate optimal protein-protein binding and/or reduce non-specific orbackground interactions. Also reagents that otherwise improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc., may be used. The mixture ofcomponents may be added in any order that provides for the requisitebinding. Various blocking and washing steps may be utilized as is knownin the art.

In a preferred embodiment, two-color competitive hybridization assaysare run. These assays can be based on traditional sandwich assays. Thebeads contain a capture sequence located on one side (upstream ordownstream) of the SNP, to capture the target sequence. Two SNPallele-specific probes, each labeled with a different fluorophore, arehybridized to the target sequence. The genotype can be obtained from aratio of the two signals, with the correct sequence generally exhibitingbetter binding. This has an advantage in that the target sequence itselfneed not be labeled. In addition, since the probes are competing, thismeans that the conditions for binding need not be optimized. Underconditions where a mismatched probe would be stably bound, a matchedprobe can still displace it. Therefore the competitive assay can providebetter discrimination under those conditions. Because many assays arecarried out in parallel, conditions cannot be optimized for every probesimultaneously. Therefore, a competitive assay system can be used tohelp compensate for non-optimal conditions for mismatch discrimination.

In a preferred embodiment, dideoxynucleotide chain-terminationsequencing is done using the compositions of the invention. In thisembodiment, a DNA polymerase is used to extend a primer usingfluorescently labeled ddNTPs. The 3′ end of the primer is locatedadjacent to the SNP site. In this way, the single base extension iscomplementary to the sequence at the SNP site. By using four differentfluorophores, one for each base, the sequence of the SNP can be deducedby comparing the four base-specific signals. This may be done in severalways. In a first embodiment, the capture probe can be extended; in thisapproach, the probe must either be synthesized 5′-3′ on the bead, orattached at the 5′ end, to provide a free 3′ end for polymeraseextension. Alternatively, a sandwich type assay can be used; in thisembodiment, the target is captured on the bead by a probe, then a primeris annealed and extended. Again, in the latter case, the target sequenceneed not be labeled. In addition, since sandwich assays require twospecific interactions, this provides increased stringency which isparticularly helpful for the analysis of complex samples.

In addition, when the target analyte and the DBL both bind to the agent,it is also possible to do detection of non-labelled target analytes viacompetition of decoding.

In a preferred embodiment, the methods of the invention are useful inarray quality control. Prior to this invention, no methods have beendescribed that provide a positive test of the performance of every probeon every array. Decoding of the array not only provides this test, italso does so by making use of the data generated during the decodingprocess itself. Therefore, no additional experimental work is required.The invention requires only a set of data analysis algorithms that canbe encoded in software.

The quality control procedure can identify a wide variety of systematicand random problems in an array. For example, random specks of dust orother contaminants might cause some sensors to give an incorrectsignal—this can be detected during decoding. The omission of one or moreagents from multiple arrays can also be detected. An advantage of thisquality control procedure is that it can be implemented immediated priorto the assay itself, and is a true functional test of each individualsensor. Therefore any problems that might occur between array assemblyand actual use can be detected. In applications where a very high levelof confidence is required, and/or there is a significant chance ofsensor failure during the experimental procedure, decoding and qualitycontrol can be conducted both before and after the actual sampleanalysis.

In a preferred embodiment, the arrays can be used to do reagent qualitycontrol. In many instances, biological macromolecules are used asreagents and must be quality controlled. For example, large sets ofoligonucleotide probes may be provided as reagents. It is typicallydifficult to perform quality control on large numbers of differentbiological macromolecules. The approach described here can be used to dothis by treating the reagents (formulated as the DBLs) as variableinstead of the arrays.

In a preferred embodiment, the methods outlined herein are used in arraycalibration. For many applications, such as mRNA quantitation, it isdesirable to have a signal that is a linear response to theconcentration of the target analyte, or, alternatively, if non-linear,to determine a relationship between concentration and signal, so thatthe concentration of the target analyte can be estimated. Accordingly,the present invention provides methods of creating calibration curves inparallel for multiple beads in an array. The calibration curves can becreated under conditions that simulate the complexity of the sample tobe analyzed. Each curve can be constructed independently of the others(e.g. for a different range of concentrations), but at the same time asall the other curves for the array. Thus, in this embodiment, thesequential decoding scheme is implemented with different concentrationsbeing used as the code “labels”, rather than different fluorophores. Inthis way, signal as a response to concentration can be measured for eachbead. This calibration can be carried out just prior to array use, sothat every probe on every array is individually calibrated as needed.

In a preferred embodiment, the methods of the invention can be used inassay development as well. Thus, for example, the methods allow theidentification of good and bad probes; as is understood by those in theart, some probes do not function well because they do not hybridizewell, or because they cross-hybridize with more than one sequence. Theseproblems are easily detected during decoding. The ability to rapidlyassess probe performance has the potential to greatly reduce the timeand expense of assay development.

Similarly, in a preferred embodiment, the methods of the invention areuseful in quantitation in assay development. Major challenges of manyassays is the ability to detect differences in analyte concentrationsbetween samples, the ability to quantitate these differences, and tomeasure absolute concentrations of analytes, all in the presence of acomplex mixture of related analytes. An example of this problem is thequantitation of a specific mRNA in the presence of total cellular mRNA.One approach that has been developed as a basis of mRNA quantitationmakes use of a multiple match and mismatch probe pairs (Lockhart et al.,1996), hereby incorporated by reference in its entirety. While thisapproach is simple, it requires relatively large numbers of probes. Inthis approach, a quantitative response to concentration is obtained byaveraging the signals from a set of different probes to the gene orsequence of interest. This is necessary because only some probes respondquantitatively, and it is not possible to predict these probes withcertainty. In the absence of prior knowledge, only the average responseof an appropriately chosen collection of probes is quantitative.However, in the present invention, this can be applied generally tonucleic acid based assays as well as other assays. In essence, theapproach is to identify the probes that respond quantitatively in aparticular assay, rather than average them with other probes. This isdone using the array calibration scheme outlined above, in whichconcentration-based codes are used. Advantages of this approach include:fewer probes are needed; the accuracy of the measurement is lessdependent on the number of probes used; and that the response of thesensors is known with a high level of certainty, since each and everysequence can be tested in an efficient manner. It is important to notethat probes that perform well are chosen empirically, which avoids thedifficulties and uncertainties of predicting probe performance,particularly in complex sequence mixtures. In contrast, in experimentsdescribed to date with ordered arrays, relatively small numbers ofsequences are checked by performing quantitative spiking experiments, inwhich a known mRNA is added to a mixture.

In a preferred embodiment, cDNA arrays are made for RNA expressionprofiling. In this embodiment, individual cDNA clones are amplified (forexample, using PCR) from cDNA libraries propagated in a host-vectorsystem. Each amplified DNA is attached to a population of beads.Different populations are mixed together, to create a collection ofbeads representing the cDNA library. The beads are arrayed, decoded asoutlined above, and used in an assay (although as outlined herein,decoding may occur after assay as well). The assay is done using RNAsamples (whole cell or mRNA) that are extracted, labeled if necessary,and hybridized to the array. Comparative analysis allows the detectionof differences in the expression levels of individual RNAs. Comparisonto an appropriate set of calibration standards allows quantification ofabsolute amounts of RNA.

The cDNA array can also be used for mapping, e.g. to mapdeletions/insertions or copy number changes in the genome, for examplefrom tumors or other tissue samples. This can be done by hybridizinggenomic DNA. Instead of cDNAs (or ESTs, etc.), other STS (sequencetagged sites), including random genomic fragments, can also be arrayedfor this purpose.

All references cited herein are incorporated by reference in theirentirety.

1-50. (canceled)
 51. A process of forming a plurality of random arraysof encoded particles on a substrate, wherein each of the arrays areformed by random placement of encoded particles, wherein differentlyencoded particles have a different label, comprising: (i) placing andconfining a group of differently encoded particles into one or moresites on a substrate having a plurality of sites, so as to form a firstconfined group of particles occupying particular sites; (ii) recordingthe positions of the encoded particles within said confined group; (iii)placing and confining an additional group of differently encodedparticles into unoccupied sites on the substrate so as to form anadditional group of confined particles occupying particular sites; (iv)recording the positions of particles within said additional confinedgroup; and repeating steps (iii) to (iv) several times so as to form aplurality of a random encoded arrays of particles.
 52. The substrate ofclaim 51 wherein the substrate comprises silicon or doped silicon. 53.The substrate of claim 51 wherein differently encoded particles havedifferent biological reagents bound thereto.
 54. The process of claim 51wherein the arrangement of the encoded particles in the sites is random.55. The process of claim 51 further comprising the step of affixing theparticles onto the substrate.
 56. A method of forming a composite arrayof randomly distributed, identifiable microspheres on a substrate havinga plurality of assay locations, comprising: (a) randomly distributing apopulation of microspheres, including subpopulations of microspherescarrying different chemical functionalities, to discrete sites within afirst assay location; (b) determining the location of saidsubpopulations of microspheres within said first assay location; (c)randomly distributing a different population of microspheres, includingsubpopulations of microspheres carrying different chemicalfunctionalities, to discrete sites within a second assay location; (d)determining the location of said subpopulations of microspheres withinsaid second assay location; and (e) repeating steps (c) to (d) as manytimes as necessary so as to form a composite array.
 57. The substrate ofclaim 56 wherein the substrate comprises silicon or doped silicon. 58.The substrate of claim 56 wherein differently encoded particles havedifferent biological reagents bound thereto.
 59. The process of claim 56wherein the arrangement of the encoded particles in the sites is random.60. The process of claim 56 further comprising the step of affixing theparticles onto the substrate.
 61. A method of forming a composite arrayof randomly distributed, identifiable microspheres on a substrate havinga plurality of assay locations, comprising: (a) providing a library ofmicrospheres comprising a plurality of sublibraries, wherein eachsublibrary further comprises a plurality of subpopulations of uniquelyidentifiable microspheres; (b) randomly distributing a first sublibrary,including subpopulations of uniquely identifiable microspheres, todiscrete sites within a first assay location; (c) determining thelocation of said subpopulations of microspheres within said first assaylocation; (d) randomly distributing a second sublibrary of microspheres,including subpopulations of uniquely identifiable microspheres, todiscrete sites within a second assay location; (e) determining thelocation of said subpopulations of microspheres within said second assaylocation; (f) repeating steps (d) to (e) with different sublibraries andassay locations multiple times so as to form a composite array.
 62. Thesubstrate of claim 61 wherein the substrate comprises silicon or dopedsilicon.
 63. The substrate of claim 61 wherein differently encodedparticles have different biological reagents bound thereto.
 64. Theprocess of claim 61 wherein the arrangement of the encoded particles inthe sites is random.
 65. The process of claim 61 further comprising thestep of affixing the particles onto the substrate.